A human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation under the standard culture condition. We examined the changes in the proliferative activity of Caco-2 cells during the differentiation, and the effect of serum factors on this spontaneous differentiation. Caco-2 cells cultured in FCS-containing media exhibited the differentiation phenotypes such as dome formation and increase of ALP activity after the 6th day of culture. Cell cycle analysis showed a marked decrease in S phase cell population during the cell differentiation. When Caco-2 cells were cultured in FCS free media, the cell population of S phase remained unchanged during culture period and the differentiation phenotypes were not induced. The addition of FCS to Caco-2 cell cultured in the absence of FCS resulted in the rapid induction of the differentiation phenotypes. These results suggest that differentiation of Caco-2 cells is closely related to the reduction of cell proliferation and requires some factors in FCS.