The effect of sodium butyrate and retinoic acid added singly or in combination on substrate-dependent growth, colonization efficiency in soft agar, and carcino-embryonic antigen (CEA) production in three human colorectal carcinoma cell lines differing in their degree of differentiation was studied. All three colon cancer cell lines regardless of their state of differentiation had their growth markedly slowed by sodium butyrate, and to a lesser extent by retinoic acid. When both agents were added together, a small synergistic inhibition of growth was noted in all the cell lines. Butyrate eliminated colony formation in soft agar in all three cell lines, however, retinoic acid only reduced colony formation in the well differentiated cell line DLD-2. Sodium butyrate was able to induce CEA production in the undifferentiated cell (MIP-101) and the moderately differentiated cells (clone D) which were previously negative for this marker. It also enhanced the baseline production of CEA in the well differentiated cells (DLD-2). Retinoic acid did not induce CEA production in clone D or MIP-101 cells, but did enhance the production of CEA in DLD-2 cells. When both retinoic acid and sodium butyrate were added together, CEA production was either additive (DLD-2) or was inhibited (clone D and MIP-101). One explanation of these results is that only well differentiated cells have functional cellular retinoic acid-binding protein (cRABP), and that certain actions of retinoic acid (inhibition of anchorage-dependent growth) are independent of the presence of cRABP.