Hypoxic proliferation of pulmonary arterial smooth muscle cells (PASMC) is mitogen dependent, but the signaling pathways mediating hypoxia-induced cell growth are not well understood. We investigated hypoxic proliferation in primary cultures from porcine pulmonary artery smooth muscle. The cells were grown in medium with or without platelet-derived growth factor (PDGF)-B, a potent smooth muscle cell mitogen. Hypoxia induced upregulation of PDGF receptor-beta expression, the primary receptor for PDGF-B. However, PDGF-B-mediated hypoxic enhancement of proliferation was abolished by pertussis toxin, indicating (1) involvement of heterotrimeric Galpha i proteins and (2) minimal effect of increased PDGF receptor expression in hypoxic enhancement of proliferation. We treated PASMC with labeled, nonhydrolyzable analogs of GTP to determine directly if GTP binding proteins were activated by hypoxia in PASMC. We show that hypoxia stimulates GTP incorporation in PASMC both in the presence and absence of PDGF-B. Serum-starved PASMC are able to increase their incorporation of GTP after only 10 min of hypoxia, and this response is not pertussis toxin sensitive. In serum-starved PASMC, we show that hypoxia stimulates incorporation of GTP into a 44-kD protein. The results show that heterotrimeric G proteins are involved in hypoxia-induced signaling in pulmonary vascular smooth muscle cells.