During the differentiation of the intestine epithelium, three cytoskeletal proteins, villin, fimbrin, and myosin I, are sequentially expressed and localized to the apical membrane. Recently, we found that in the adult mouse and human, three fimbrin isoforms are expressed in a cell specific manner. I-fimbrin is expressed by intestine and kidney epithelial cells, L-fimbrin is expressed by leukocytes and many tumors, while T-fimbrin is expressed by various cells and tissues. Because non-intestinal isoforms of fimbrin could be expressed early in development, the expression of fimbrin isoforms during days 10.5 to 16.5 of intestine development was investigated. By immunofluorescence microscopy, T-fimbrin was detected in the early stages of intestinal epithelial cell differentiation until day 14.5 and was localized predominantly at the apical surface. L-fimbrin was also detected during this period but it was localized to the basal surface of the epithelium instead of the apical surface. By day 16.5 no L or T-fimbrin was detected in the epithelium. I-fimbrin was first detected at day 14.5 and a brush border-like apical localization pattern was seen by day 16.5. Unlike the intestinal cells, the visceral endoderm expressed I, L, and T-fimbrin throughout the period examined, with the level of I-fimbrin increasing as time progresses. L-fimbrin was more evident at the earlier stage than at the later stage of the development. Collectively, these results suggest that three fimbrin isoforms play different roles during epithelial cell differentiation. T- and I-fimbrin expression could be critical for the formation and extension of the microvilli whereas L-fimbrin may play a role in controlling cell adhesion.