We have purified and characterized the 50 kd activator protein 2 (AP-2), another enhancer-binding protein interacting with the human metallothionein IIA (hMT-IIA) gene control region. Purified AP-2 activates transcription in vitro from a hybrid promoter containing hMT-IIA upstream sequences. AP-2 also recognizes control elements of the human growth hormone, c-myc, and H-2Kb genes, and the SV40 and bovine papilloma virus enhancers. Multiple synthetic copies of the hMT-IIA high-affinity AP-2 binding site can act as efficient, cell-type-specific enhancer elements; their activity increases after treatment of cells with phorbol ester or cAMP-elevating agents. In contrast, a synthetic enhancer recognized by factor AP-1 is activated only by phorbol ester. AP-2 appears to mediate transcriptional activation in response to two different signal-transduction pathways, one involving the phorbol-ester- and diacylglycerol-activated protein kinase C, the other involving cAMP-dependent protein kinase A.