Chronic hypoxic exposure induces marked remodeling in the adventitial compartment of the pulmonary artery, which is due, at least in part, to increased proliferation of the resident fibroblast populations. We have demonstrated that hypoxia can stimulate the proliferation of adventitial fibroblasts through the activation of protein kinase C (PKC) and extracellular signal-regulated kinases (ERKs) 1/2 pathways and that those proliferative responses are greater in fibroblasts derived from chronically hypoxic animals than those from the controls. Mitogen-activated protein kinase phosphatase 1 (MKP1), an important terminator of ERK activation, has been identified as a hypoxia-inducible gene. However, the role of MKP1 in the hypoxia-induced proliferation of fibroblasts is unknown. Since hypoxia activates the PKCζ isozyme in many cell types, we hypothesized that PKCζ will control the hypoxic proliferation of vascular fibroblasts through the regulation of MKP1, and thus ERK1/2, and that this PKCζ-mediated mechanism will be different in fibroblasts from control animals vs chronically hypoxic animals. Using fibroblasts isolated from the pulmonary artery adventitia of neonatal control and chronically hypoxic calves, we found that, hypoxia, in the absence of any comitogens, induced the activation of PKCζ. The treatment of control cells with either a myristoylated pseudosubstrate peptide inhibitor or antisense oligonucleotides of PKCζ induced a marked increase in hypoxia-induced proliferation, significant up-regulation of ERK1/2 activation, and the blockade of hypoxia-stimulated increases in MKP1 levels. In contrast, in fibroblasts from chronically hypoxic calves, PKCζ inhibition blocked hypoxia-induced proliferation as well as ERK1/2 activation. MKP1 was not up-regulated in these cells under hypoxic conditions. These results demonstrate that chronic hypoxic exposure induces changes in the downstream targets of PKCζ in vascular adventitial fibroblasts. In normal fibroblasts, PKCζ-mediated MKP1 activation acts to limit cell proliferation. However, following chronic hypoxic exposure, PKCζ-regulated activation of MKP1 is lost, resulting in fibroblasts with heightened proliferative capabilities.