Significant diurnal variation was observed in ovarian ascorbic acid (OAA) content in rats rendered pseudopregnant by injections of PMS and HCG. Exposing the rats to varying periods of light throughout the day did not change the diurnal pattern. Since the level of OAA is an important variable in the assay of LH by the OAA depletion method of Parlow (1, 2), the time of day that assays are performed appears to be critical. (Endocrinology 74: 493,1964) P (1, 2) described a convenient and sensitive method for assaying luteinizing hormone (LH) by measuring the depletion of ovarian ascorbic acid (OAA) in immature pseudopregnant rats following an intravenous injection of LH. Sakiz and Guillemin (3) have pointed out some of the variables encountered with this method, such as the strain of rats used, priming of assay animals, and methods of expressing the results of the assay. We have made an additional observation on the variation in OAA content in pseudopregnant rats during a 24-hr period. This variation appears to be an important variable in the procedure described by Parlow. Materials and Methods The Holtzman strain of the Sprague-Dawley rat was used throughout the study. Twenty-fiveday-old female rats were injected with 100 IU of pregnant mare's serum (PMS) and 56-64 hr later with 50 IU of human chorionic gonadotropin (HCG). Seven days following the HCG injection the animals were used for study. In 2 of 3 experiments, the ascorbic acid content of both ovaries was determined in 10 rats every 3 hr starting at 8:00 AM and continuing for 27 hr, while in a third the experiment was continued for only 21 hr. The 3 experiments were performed at approximately 2-month intervals. Experiments 1 through 3 were conducted on Received August 1, 1963. 1 Work performed during tenure as a Postdoctoral Fellow supported by the Population Council, Rockefeller Institute, New York. Present address: The Department of Obstetrics and Gynecology, Medical School, Tohoku University, Sendai, Japan. January 24, 1963, March 4, 1963, and May 10, 1963, respectively. The rats were housed in an air-conditioned room for at least 10 days prior to use and allowed constant access to food throughout the study. In routine studies the rats were exposed to 14 hr of light and 10 hr of darkness per day. In order to determine the acute effects of light on OAA content, an experiment was performed with some rats exposed to constant light for 24 hr and others to only 10 hr of light during the same interval. The animals were sacrificed by dislocation of the cervical vertebrae, the ovaries were removed, and the ascorbic acid content was determined by a modification of the method of Mindlin and Butler (4). Only those ovaries weighing 80 mg or more were used for the study.