Allergic proctocolitis is a major cause of rectal bleeding in infants, but its clinical features and laboratory results are often nonspecific. Our previous retrospective study demonstrated that large numbers of eosinophils in colonic mucosal biopsy specimens were highly associated with cases of allergic proctocolitis. Therefore, we prospectively examined 60 colonic mucosal biopsy specimens from the same sites (4, 8, and 12 cm from the anal verge) in 20 infants with clinically confirmed allergic proctocolitis to validate this morphologic feature, to characterize its distribution, and to correlate these data with the clinical information. The patients (age range, 4 to 304 days) were fed breast milk or a variety of formulas and all presented with rectal bleeding. Sigmoidoscopic examination was abnormal in 19 cases, typically characterized by focal areas of mucosal erythema. The major histologic finding was a strikingly focal increase in the number of eosinophils in all mucosal compartments, with a predilection to aggregate in close association with lymphoid nodules. Eosinophilic infiltration varied not only between biopsies at different sites, but also within individual biopsy specimens. Only 12 of 20 patients (60%) had all three of their biopsy specimens categorized as abnormal; in the remainder, only one (four patients) or two (four patients) of the three biopsy specimens were abnormal. The average number of eosinophils per high-power field of lamina propria for all cases was 15.6. No significant correlation was identified between the number of eosinophils in the mucosa and the patient's age, length of illness, endoscopic appearance, or type of inciting formula. In summary, eosinophils appear to be an excellent marker for infantile allergic proctocolitis. Given the focal distribution of the eosinophils, multiple mucosal biopsy specimens should be obtained and several levels of each examined to confirm the diagnosis.