The endothelial isoform of nitric oxide synthase (ec-NOS) is targeted to the particulate subcellular fraction by means of N-terminal myristoylation. However, the association of ecNOS with the particulate subcellular fraction appears to be dynamically regulated, in that agonist treatment of endothelial cells induces translocation of the enzyme from membrane to cytosol (Michel, T., Li, G., and Busconi, L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6252-6255). cDNA encoding wild-type and myristoylation-deficient mutant (myr-) ecNOS was transcribed and translated in vitro, and we found that the recombinant wild-type but not the myr- mutant protein undergoes myristoylation and is able to associate with biological membranes prepared from diverse cell sources. Treatment of these cell membranes with heat or with trypsin did not affect their ability subsequently to serve as acceptor membranes for the wild-type recombinant enzyme. The wild-type ecNOS, but not the myr- mutant, is able to form stable associations with phospholipid liposomes. We also explored the possibility that a polybasic domain within the ecNOS protein might serve as a secondary structural determinant for ecNOS membrane association and constructed truncation mutants that flank a polybasic domain present in the ecNOS. These truncation mutants, transcribed and translated in vitro or transfected into COS-7 cells, undergo myristoylation and are able to associate with biological membranes in a fashion indistinguishable from the wild type ecNOS. Taken together, these results indicate that ecNOS binding to biological membranes is dependent upon interactions of the N-terminal myristoyl moiety of ecNOS with lipid components of the membrane, and this association does not require a specific membrane protein functioning as a myristate receptor nor the presence of a polybasic domain within the ecNOS.