PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a lipid phosphatase that functions as a negative regulator of the phosphoinositide-3-kinase (PI3K) pathway. The present study sought to examine in depth the interaction between PTEN and eNOS activity. Co-expression of eNOS and PTEN in COS-7 cells significantly decreased NO production compared to eNOS alone, while co-expression of eNOS and the dominant negative mutant PTEN(C124A) significantly increased NO production. Upon examination of the putative eNOS phosphorylation sites, phosphorylation of S116, T497, S617, S635 and S1179 was decreased by PTEN co-expression, while the dominant negative PTEN(C124A) produced an increase in phosphorylation of all sites except S116 and S635. A myristoylation-deficient eNOS construct with little dependence on phosphorylation state (G2AeNOS) was not significantly affected by co-expression with either PTEN or PTEN(C124A). Likewise, an eNOS construct with a triple phospho-null mutation (S617A, S635A and S1179A) was also unaffected by co-expression with either PTEN or PTEN(C124A). Purified PTEN or PTEN(C124A) failed to interact with purified eNOS in vitro, arguing against a direct interaction between PTEN and eNOS. When the PTEN constructs were expressed in human aortic endothelial cells (HAECs), PTEN significantly decreased NO production and PTEN(C124A) increased it, and both S617 and S1179 were altered by co-expression with the PTEN constructs. Increased expression of PTEN in endothelial cells did not influence superoxide production. We conclude that PTEN is a regulator of eNOS function both when expressed in COS-7 cells and in human endothelial cells, and does so via its effects on the PI3K/Akt pathway.