The characteristics of adult bovine adrenocortical cells were studied throughout their life span of 55-65 generations in monolayer culture. Over this period, the cells maintained the capacity to synthesize steroids when tested with repeated maximal doses of ACTH, prostaglandin E1, monobutyryl cAMP, or cholera toxin. Prostaglandin E1 stimulated cAMP production and steroidogenesis, and inhibited DNA synthesis, as measured by incorporation of [3H]thymidine, with dose-response characteristics that did not vary over the first 50 generations in culture. In contrast, the maximal rate of cAMP production stimulated by ACTH declined exponentially at a rate of 7% per generation. In primary and secondary cultures, ACTH stimulated steroidogenesis maximally and inhibited [3H]thymidine incorporation into DNA completely at a half-maximal effective concentration (ED50) of 0.08 nM which was two orders of magnitude less than the ED50 of 8 nM for stimulation of cAMP production. As the ACTH-stimulated maximal rate of cAMP production fell with increasing generation number, the ED50 for ACTH stimulation of steroidogenesis and inhibition of DNA synthesis increased. From about the 20th generation onward, the ability of ACTH to inhibit DNA synthesis maximally declined so that by the 40th generation, cells were completely resistant to the growth-inhibitory effects of ACTH. High-dose ACTH continued, however, to stimulate steroid production maximally over the 50 generations studied. In late passage cells, the ED50 for ACTH stimulation of steroidogenesis was 8 nM, identical to that for cAMP production. Although ACTH-stimulated cAMP production was related to both stimulation of steroidogenesis and inhibition of DNA synthesis, higher cAMP levels appeared required for inhibition of DNA synthesis than for stimulation of steroidogenesis. Mitogenic responses to fibroblast growth factor and to angiotensin II were retained throughout long term growth in culture. The progressive loss of ACTH-responsiveness was specific and a function of aging of bovine adrenocortical cells in culture.