The signaling mechanisms in vasculogenesis and/or angiogenesis remain poorly understood, limiting the ability to regulate growth of new blood vessels in vitro and in vivo. Cultured human lung microvascular endothelial cells (HLMVEC) align into tubular networks in the 3-dimensional matrix, Matrigel. Overexpression of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates the ERK, JNK and p38 pathways inhibited network formation of these cells. Adenoviral-mediated over-expression of recombinant MKP-3 (a dual specificity phosphatase that specifically inactivates the ERK pathway) and dominant negative or constitutively active MEK did not attenuate network formation in Matrigel as compared to negative controls. This result suggested that the ERK pathway may not be essential for tube assembly which conclusion was supported by the action of specific MEK inhibitor PD 184352 which also did not alter network formation. Inhibition of the JNK pathway using SP600125 or L-JNKI-1 blocked network formation while the p38 MAPK blocker SB 203580 slightly enhanced it. Inhibition of JNK also attenuated the number of small vessel branches in the developing chick chorioallantoic membrane. Our results demonstrate a specific role for the JNK pathway in network formation of human lung endothelial cells in vitro while confirming that it is essential for the formation of new vessels in vivo.