The transactivating function of the c-Jun proto-oncogene component of the AP-1 transcription factor is acutely regulated by a wide variety of cellular signals via modulation of phosphorylation of two serines (63 and 73). The viral oncoprotein, v-Jun, while containing homologous serines, is not phosphorylated in cells. A novel family of stress-activated protein kinases (SAPKs), also termed Jun N-terminal domain kinases (JNKs), are responsible for mediating S63/73 phosphorylation in response to a variety of cellular stimuli including tumor necrosis factor-alpha, heat stress and u.v. light. The p54 alpha 1, alpha 2, p54 beta and p46 beta SAPKs are shown to bind directly to c-Jun but not to v-Jun, with an absolute requirement for c-Jun amino acids 31-47, a region deleted in v-Jun. Inactive SAPKs tightly bind c-Jun in resting cells and may be a manifestation of the 'delta' inhibitor, a previously described repressor of c-Jun function.