Pairing of metacercariae or adults of Leucochloridiomorpha constantiae was studied in the bursa of Fabricius of the domestic chick, on the chick chorioallantois, and in vitro at 37.5 C in agar cultures containing a Locke's overlay. Worm pairs were observed in all sites at various intervals from 0.5 hr to 8 days postinoculation. In vitro and chorioallantoic observations indicate pairing is worm-mediated and not dependent on factors associated with the bursa. Observations on noncontact pairing indicate chemical attractants produced by worms rather than thigmotaxis may be involved. Transection of worms more adversely affected pairing of posteriorthan anterior-half worms with whole ones in vitro suggesting that factors associated with pairing are mostly in the worm's anterior region. Limited observations are available on the pairing of hermaphroditic digenetic trematodes. Sogandares-Beral (1966) observed pairing and development of patent infections of Paragonimus kellicotti in double-worm infections in the domestic cat and suggested chemical attractants produced by worms may be involved in pairing. Recently, Fried and Harris (1971) reported pairing in adult Leucochloridiomorpha constantiae in the bursa of Fabricius of the domestic chick and reproductive advantages observed in doubleas compared with singleworm infections were presumably related to factors associated with worm-pairing and crossinsemination. The purpose of this study was to extend observations on worm-pairing of L. constantiae in the chick and to study this phenomenon in vitro and on the chick chorioallantois. MATERIALS AND METHODS L. constantiae metacercariae were obtained as described previously (Fried and Harris, 1971) and 6to 14-day-old flukes were raised in 1to 7-dayold domestic chicks using methods noted in the aforementioned paper and in Allison (1943). Worms were washed rapidly in 3 changes of sterile Locke's (Paul, 1960) prior to use in the following experiments. In vitro studies were made in 5-cm sterile plastic petri dishes containing a substrate of 8 to 10 ml 0.85% Oxoid ion agar, No. 2 (Consolidated Lab. Inc., Chicago, Ill.) and a 5to 6-ml overlay of sterile Locke's. Using aseptic transfer techniques Received for publication 2 August 1971. * Supported in part by a research grant from the Lafayette College Committee on Advanced Study and Research. either 2 metacercariae or 2 adults were placed on the agar 20 mm apart and maintained at 37.5 C on a slide warmer (Fisher Scientific Co., Pittsburgh, Pa.). To determine the effects of transection on worm-pairing, metacercariae and adults were transected with a microscalpel through the midacetabular region and anterioror posteriorhalf metacercariae were placed in cultures with whole larvae. Whole and transected adults were prepared similarly. In vitro controls consisted of a metacercaria or an adult placed in cultures with another worm of similar age boiled in water for 5 min or frozen at -20 C for 1 to 2 days. Live organisms were also matched with either filter paper or charcoal of approximate worm size. The distance between 2 organisms or an organism and an inert control was measured in mm 0.5, 1, 2, 3, 4, and 24 hr postinoculation and combinations in contact or within 5 mm of each other were scored as paired. Pairing on the chorioallantois was studied in 83 8to 12-day-old fertile hens' eggs prepared according to Zwilling (1959). Each chorioallantois was exposed to either 2 metacercariae or 2 adults through separate windows cut 20 mm apart in the egg shell. Worms were maintained on membranes at 37.5 C from 0.5 to 192 hr postinoculation and at necropsy the egg shell surrounding windows was removed and the chorioallantois searched for organisms in contact or within 5 mm of each other. Since recovery of 2 worms from an egg in doubleworm exposure studies was difficult, 17 additional eggs each exposed to 3 to 6 metacercariae through a single window were examined 1 to 8 days postinoculation to observe worm-pairing or clustering (3 or more worms in contact). Pairing in the bursa of Fabricius was studied in 104 1to 7-day-old chicks each exposed to either 2 metacercariae or 2 adults. Chicks were necropsied at various intervals from 0.5 to 120 hr postexposure and the bursa removed and inverted in Ringer's. Organisms in contact or within 2 mm of each other on the same or adjacent bursal folds were scored as paired.