Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide (.NO). A baculovirus overexpression system has been developed for a constitutive NOS isoform, cloned originally from rat cerebellum (B-NOS). Recombinant virus was used at a multiplicity of infection of 5 to infect Spodoptera frugiperda cells in culture, and NOS was expressed to 10% of the total soluble protein at 48 h postinfection. In order to express catalytically active enzyme, it was necessary to supplement the culture media with hemin. This increased the activity of the enzyme 7-fold. A two column affinity purification was developed for the recombinant enzyme, which gave homogeneous protein that migrated at 150 kDa on a denaturing polyacrylamide gel. A Km for L-arginine was determined to be 2.0 +/- 0.4 microM. As isolated, recombinant B-NOS exhibited a Soret maximum at 402 nm, which shifted to 394 nm in the presence of L-arginine. The Soret maximum of the reduced enzyme in the presence of CO was 444 nm. Initial rate steady-state kinetic analysis of the recombinant B-NOS showed evidence of substrate inhibition by L-arginine, which could also be seen in a partially purified preparation of B-NOS from rat cerebella. This substrate inhibition was not observed with the inducible isoform of NOS, purified from immunostimulated murine macrophages. A C415H mutant was overexpressed and purified using the same conditions established for the wild-type recombinant B-NOS. This C415H mutant exhibited no activity and did not bind heme, providing the first experimental evidence to support previously reported primary amino acid comparisons which suggest that C415 provides the coordinating thiolate to the heme moiety in B-NOS.