Pulmonary artery (PA) smooth muscle cell (SMC) proliferation is an important contributor to the vascular remodeling that occurs in chronic hypoxic pulmonary hypertension. The earliest SMC proliferative changes in response to hypoxia occur in the outer media. We tested the hypothesis that the pattern of hypoxia-induced PA SMC proliferation observed in vivo is determined at least in part by intrinsic differences in proliferative response of SMC isolated from different medial layers to relevant peptide mitogens and hypoxia. Adult bovine PA SMCs were isolated at the same proximal site from the middle (layer 2) and outer (layer 3) media. In response to maximal serum stimulation, PA SMCs from the outer media grew faster than cells from the middle media. The outer medial cells also had increased responsiveness to multiple peptide mitogens (IGF-I, PDGF-BB, bFGF, and EGF). Because protein kinase C (PKC), a key pro-proliferative signal transduction pathway, has been shown to play an important role in this type of global increase in growth, responsiveness to a direct cell-permeable activator of PKC (PMA, phorbol 12-myristate 13-acetate) was then measured. PA SMCs from the outer media had greater DNA synthesis in response to selective PKC activation than middle medial cells. Since activation of this kinase is a requisite step for PA SMCs to proliferate in response to hypoxia, the hypoxic growth potential of cells from the middle and outer media was then compared. SMCs from the outer media had an augmented proliferative response to hypoxia compared with those from the middle media. These data suggested an important role for PKC in the enhanced growth of PA SMCs from the outer media. Therefore, whole cellular activity, expression, and hypoxia-induced activation of PKC were measured in both subpopulations of PA SMCs. Outer medial cells had greater total cellular activity, expression, and hypoxia-induced activation of PKC (and the alpha isozyme in particular) than cells isolated from the middle media. These findings support the concept that heterogeneity in growth capacity of PA SMCs exists within the bovine PA media, that these intrinsic differences in growth govern, at least in part, the pattern of abnormal SMC proliferation observed in vivo, and that the PKC pathway (and PKC-alpha in particular) is likely an important determinant of the subpopulation-specific differences found.