HeLa S3 cells, a clonal strain adapted for growth in suspension culture, contain adenylate cyclase activity that is stimulated by glucagon, prostaglandin E(1), and epinephrine. Total enzymatic activity and response to hormones is increased as a result of these cells being in stationary culture for several days. The parental strain of HeLa cells normally grown in stationary culture shows even greater adenylate cyclase activity. Hepatoma (HTC) cells grown in suspension culture have no detectable adenylate cyclase activity, but when grown in stationary culture they contain low, but detectable, amounts of adenylate cyclase, which is stimulated by glucagon, epinephrine, or prostaglandins. Chang's liver cells, both suspension and stationary culture, contain relatively high levels of adenylate cyclase that is stimulated by epinephrine or prostaglandin E(1), with differences in activity depending upon culture conditions. Adenylate cyclase of 3T3 and L929 mouse fibroblasts respond to catecholamines, as well as to prostaglandin E(1), but not to glucagon. Characteristic increases in basal activity of adenylate cyclase and in activity in the presence of hormone occurred as cell density increased during stationary culture of certain cell lines, but do not occur with fluoride ion present. In addition to the influence of growth conditions and rate or phase of cell growth on cyclase activity and hormonal response, the cultured cell lines frequently showed marked differences in activity from one another; malignant cells generally exhibited less activity. It is postulated that adenylate cyclase activity may be enhanced by cell to surface contact or cell to cell interaction and that this phenomenon may represent a form of self regulation or modulation of hormonal response by a tissue.