Microvillus membranes (MVM) were isolated from newborn and adult New Zealand rabbit small intestine. The isolation procedure provided a mean enrichment of 25 +/- 4 for sucrase activity in adult preparations and of 27 +/- 3 for lactase activity in newborn preparations. These purified MVM were incubated with increasing concentrations of 125I-labeled cholera toxin (CT). 125I-CT binding to adult MVM reached saturation at 6.4 X 10(-9) M; in contrast 125I-CT binding to newborn MVM did not reach saturation but instead continued to increase with increasing 125I-CT concentrations. Scatchard plot analysis of adult data supported the existence of a single binding site (Kd = 1.2 +/- 0.2 X 10(-9) M); analysis of newborn data, however, suggested the existence of additional binding sites, as 125I-CT binding to newborn MVM was inhibited by preincubation with unlabeled CT. These results show that CT binding to both preparations is quantitatively different and is higher in newborn preparations. This difference may be accounted for by the existence of additional binding sites in newborn MVM preparations in contrast to the presence of only the unique receptor previously reported in adult MVM preparations.