There is no standard procedure in regard to the type or feeding pattern of prestudy diet in studies on determinations of iron bioavailability. The objective of this study was to determine the influence of meal frequency on the absorptive capacity of mucosal cells. Male Wistar rats were assigned to three groups of ten rats each and fed 12 g of diet daily for three days. The food allotment was provided in either one meal (M1) daily (70 mg Fe / kg diet), four meals (M4) daily (70 mg Fe / kg diet), or a single meal for one day (210 mg Fe / kg diet) followed by single low-iron meals (9.4 mg Fe / kg diet) for two days (MX). The rats were then deprived of food for 18 hours, administered 59Fe by gavage, and killed ten hours later. Absorption was estimated as the 59Fe retained in carcass without the gastrointestinal tract. Mucosal cells from the duodenum were isolated by mechanical vibration and mucosal ferritin determined by an immunoassay method. The effect of meal frequency on absorption efficiency of 59Fe was significant and independent of liver iron stores. Absorption of 59Fe dose was higher in rats fed four meals daily (42.2 ± 5.2%) than those fed one meal daily (27.1 ± 2.7%), but rats fed a single meal for one day then iron-deficient meals for two days absorbed significantly more 59Fe (64.8 ± 4.8%) than did rats in the other groups. These data indicate that the frequency of iron consumed in a short-term period alter the efficiency of iron absorption in rats. Iron status of mucosal cells alone, as determined by mucosal ferritin, cannot explain the variations in iron absorption.