Endothelial cells (ECs) from the macrovascular (ie, pulmonary artery) and microvascular (ie, pulmonary capillary) beds are phenotypically and functionally distinct. Consistent with this, pulmonary artery ECs (PAECs) within monocrotaline-treated, hypertensive adult rat lungs interact with an extracellular matrix enriched with tenascin-C, whereas pulmonary microvascular ECs (MVECs) do not. This finding suggests that tenascin-C might contribute to EC heterogeneity within the hypertensive lung. To test this, tenascin-C expression was compared in adult rat PAECs and MVECs cultured on tissue culture plastic, chosen to represent an injured tissue microenvironment. Similar to our in vivo findings, tenascin-C expression was significantly greater in cultured PAECs than in MVECs. Since activated focal adhesion kinase (FAK) is required for expression of Prx1, a homeobox gene transcription factor essential for tenascin-C expression, we next examined and found that levels of activated FAK and Prx1 are also higher in tenascin-C–producing PAECs than in MVECs. Furthermore, inhibition of FAK activity not only reduced Prx1 expression in PAECs, but this also resulted in phenotypic change underscored by disruption of cytocortical F-actin. Since tenascin-C modulates F-actin dynamics through suppression of RhoA, (a small guanosine triphosphatase required for F-actin stress fiber formation), we reasoned that RhoA activity and F-actin distribution would differ in PAECs vs MVECs. Indeed, whereas PAECs possessed lower levels of RhoA activity and exhibited cytocortical F-actin, MVECs had higher levels of RhoA activity and displayed prominent F-actin stress fibers. Finally, to determine whether interactions with tenascin-C directly contribute to pulmonary EC heterogeneity, we cultivated tenascin-C–deficient MVECs on exogenous tenascin-C; this treatment suppressed RhoA activity, an event that was associated with loss of F-actin stress fibers in favor of cytocortical F-actin. Collectively, these data indicate that pulmonary EC heterogeneity observed within the hypertensive lung may depend, in part, on the nature of the surrounding extracellular matrix.