The expression of carcinoembryonic antigen (CEA) is thought to be involved in homotypic adhesion and has been associated with the progression of human colon carcinomas (HCC) to the metastatic state. Three cell lines established from surgical specimens of Dukes' stage D (KM20) or Dukes' stage B (KM12C, KM12SM) with high and low preoperative CEA serum levels, respectively, were studied subsequent to growth in culture, in the subcutis (ectopic) or cecal wall (orthotopic) of nude mice. In all cell lines, CEA expression was higher in cecal tumors than in subcutaneous lesions. The degree of differentiation and CEA expression by HCC growing in the cecal wall of nude mice correlated with the pathological diagnosis and preoperative CEA level of the original patients. To better understand the regulation of CEA expression, the HCC cells were grown in culture as sparse and confluent monolayers or as multicell spheroids. The CEA expression level increased in all three cell lines growing as confluent monolayers and was highest in multicell spheroids. Treatment of sparse monolayer cultures of KM12SM cells with mitomycin-C inhibited cell division and was associated with higher production of CEA protein. Moreover, conditioned media from confluent monolayer cultures or from spheroids up-regulated CEA production in sparse monolayer cells. These data show that CEA expression in HCC cells may be regulated by cell density and by factors from the organ environment.