Squamous-cell carcinoma antigen (SCC antigen), formerly referred to as TA-4, is closely related to the grade of differentiation. Immunohistochemical studies have demonstrated that SCC antigen is also increased in the keratinizing or large-cell nonkeratinizing type of squamous-cell carcinoma but not in the small cell type. On the other hand, the appearance rate of SCC antigen in the blood circulation is almost the same in these three types of squamous-cell carcinoma. The present study was conducted to clarify the discrepancy in the production and release of this tumor marker using a squamous-cell carcinoma cell line, SKG-IIIa. SKG-IIIa cells were treated with 10 microM 5-azacytidine, a potent hypomethylating agent, to obtain several sublines with different behavior in the production of SCC antigen, and cloned by a limiting dilution technique. One subline (B-5) released significantly greater amounts of SCC antigen into the incubation medium as compared with other sublines (A-5). In in vivo studies, groups of nude mice received subcutaneous injections of the A-5 or B-5 subline, and the serum SCC antigen levels were determined after the animals exhibited palpable tumors. The serum levels of SCC antigen were significantly higher in the animals inoculated with the B-5 cells than in those inoculated with the A-5 cells. On the other hand, flow-cytometric analysis and immunohistochemical studies using a polyclonal antibody revealed that the intracellular contents of SCC antigen were greater in the A-5 cells than in the B-5 cells. Radioautography was performed using a 125I-labeled monoclonal antibody specific against the acidic fraction of this marker, a dominant form released outside the cells, which revealed that the production of the acidic fraction was somewhat greater in the B-5 cells than in the A-5 cells. These results suggest that the production and release of SCC antigen are different phenomena in squamous-cell carcinoma and that the release of SCC antigen is likely influenced by the production of the acidic fraction.