The levels of control involved in the regulation of expression of the carcinoembryonic antigen (CEA) gene family members were investigated using cultured Caco-2 cells that differentiated after confluence as characterized by the production of polarized monolayers and the subsequent appearance of domes. Three transcripts representing CEA (3.0 and 3.5 kilobases) and nonspecific cross-reacting antigen (NCA) (2.6 kilobases) were detected in Northern analyses of mRNA preparations of such cells when probed with human CEA cDNA, albeit at different levels. The major CEA 3.0-kilobase transcript increased 3-fold over an 11-day culture period after confluence, whereas the NCA transcript increased 25-fold over the same time period. gamma-interferon treatment enhanced CEA mRNA levels 32-fold and NCA mRNA levels 53-fold in Caco-2 monolayers 11 days after confluence. The NCA gene thus appears to be regulated by a mechanism different from that of CEA. During gamma-interferon treatment, the normal increase in Caco-2 dome formation with time in culture was increased further by a factor of 2. Over the 13-day time span for Caco-2 cultures, CEA protein levels increased 7-fold, NCA (Mr 48,000) protein 5-fold, while gamma-interferon treatment augmented CEA 18.5-fold further and NCA 20-fold. In 2 other colon carcinoma cell lines, SW1222 and T84, which are differentiated in culture to varying degrees, little if any changes were seen in CEA and NCA mRNA and protein levels in pre- versus postconfluent cultures. Both cell lines, however, responded to gamma-interferon treatment by increases in CEA and NCA mRNA levels and, in some cases, disproportionate increases in the corresponding proteins. The lack of direct proportionality between mRNA and protein expression suggests that, as observed in human colon carcinoma and adjacent normal tissue, both transcriptional and post-transcriptional control mechanisms regulate CEA gene family member expression in colon carcinoma cells.