The human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation during growth and expresses a number of brush-border membrane-associated hydrolases typical of a differentiated phenotype. Among these is the enzyme alkaline phosphatase, which is frequently used as a marker of cell differentiation in colon cancer cells. Since the biochemical processes regulating the expression of alkaline phosphatase during cell differentiation are only poorly understood, we examined the biosynthesis and processing of alkaline phosphatases in undifferentiated (0-day confluent) and differentiated (14-day confluent) Caco-2 cells. It was found that both cell phenotypes expressed a single, heat-labile intestinal-like enzyme, which undergoes similar posttranslational processing and glycosylation. Although the rate of enzyme synthesis and alkaline phosphatase messenger ribonucleic acid was 5–6-fold higher in differentiated cells, the degradation rates in both cell types were similar with a half-life of approximately 10 days. These results suggest that the increase in alkaline phosphatase activity during Caco-2 cell differentiation is caused by changes in the synthetic rate and that the low turnover rates facilitate accumulation of the enzyme. Furthermore, these studies demonstrate that Caco-2 cells are useful for examining the molecular and biochemical events involved in the differentiation of the small intestinal epithelium.