The characteristics of the sensitization and desensitization of superfused, rat dissociated adrenal fasciculata cells were examined. The dynamic output of steroids and cyclic AMP was determined following pulsed treatment with various agonists. Repeated doses of identical small amounts of ACTH or ACTH gave gradually increasing responses which were maximal after 3-4 injections, but then desensitized the adrenal cells. An initial dose of 2.5 X 10(-11) moles of ACTH, itself insufficient to stimulate steroidogenesis in this system, had the effect of priming the cells, which showed an enhanced initial response and achieved maximum responsiveness on the second injection of 4 X 10(-13) moles ACTH. Thereafter, although the cells exhibited a diminishing steroid output, a dose at the end of the experiment of 8 X 10(-12) moles ACTH restored the maximum responsiveness, and demonstrated that cell loss or death could not account for the desensitization effect. Only a sensitization of the cells was observed following repeated doses of 5 X 10(-6) moles cyclic AMP. Since no desensitization effect was discernible for this agonist, it was concluded that in this system the lesion giving rise to the desensitization effect occurred prior to the adenylate cyclase catalytic unit for the generation of cyclic AMP within the cell and the receptor-nucleotide-regulatory protein complex is thus implicated in the desensitization mechanism for adrenal steroidogenesis. The studies demonstrate the exquisite sensitivity of adrenal cells to the desensitizing effects of even brief intermittent pulses of ACTH.