Endothelial cells perform a number of important functions including release of vasodilators, control of the coagulation cascade, and restriction of solutes and fluid from the extravascular space. Regulation of fluid balance is of particular importance in the microcirculation of the lung where the loss of endothelial barrier function can lead to alveolar flooding and life-threatening hypoxemia. Significant heterogeneity exists between endothelial cells lining the microcirculation and cells from larger pulmonary arteries, however, and these differences may be relevant in restoring barrier function following vascular injury. Using well defined populations of rat endothelial cells harvested from the pulmonary microcirculation (PMVEC) and from larger pulmonary arteries (PAEC), we compared their growth characteristics in low serum conditions. Withdrawal of serum inhibited proliferation and induced G₀/G₁ arrest in PAEC whereas PMVEC failed to undergo G₀/G₁ arrest and continued to proliferate. Consistent with this observation, PMVEC had an increased cdk4 and cdk2 kinase activity with hyperphosphorylated (inactive) retinoblastoma (Rb) relative to PAEC as well as a 3-fold increase in cyclin D1 protein levels; overexpression of the cdk inhibitors p21^Cip1/Waf1 and p27^Kip1 induced G₀/G₁ arrest. While serum withdrawal failed to induce G₀/G₁ arrest in non-confluent PMVEC, confluence was associated with hypophosphorylated Rb and growth-arrest; loss of confluence led to resumption of growth. These data suggest that non-confluent PMVEC continue to proliferate independent of growth factors. This proliferative characteristic may be important in restoring confluence (and barrier function) in the pulmonary microcirculation following endothelial injury.