Human cyclooxygenase-2 (hCox-2) is a key enzyme in the biosynthesis of prostaglandins and the target of nonsteroidal anti-inflammatory drugs. Recombinant hCox-2 overexpressed in a vaccinia virus (VV)-COS-7 system comprises two glycoforms. Removal of the N-glycosylation consensus sequence at Asn580 (N580Q and S582A mutants) resulted in the expression of protein comprising a single glycoform, consistent with the partial N-glycosylation at this site in the wild-type (WT) enzyme. The specific cyclooxygenase activities of the purified WT and N580Q mutant were equivalent (40 +/- 3 mumol O2/min/mg) and titrations with diclofenac showed no difference in inhibitor sensitivities of WT and both mutants. Results of the expression of WT and N580Q hCox-2 in a Drosophila S2 cell system were also consistent with the N-glycosylation at this site, but low levels of activity were obtained. High levels of N-glycosylation heterogeneity are observed in hCox-2 expressed using recombinant baculovirus (BV) in Sf9 cells. Expression of a double N-glycosylation site mutant in Sf9 cells, N580Q/N592Q, resulted in a decrease in glycosylation but no clear decrease in heterogeneity, indicating that the high degree of N-glycosylation heterogeneity observed with the BV-Sf9 system is not due to partial glycosylation of both Asn580 and Asn592. N-linked oligosaccharide profiling of purified VV and BV WT and S582A mutant hCox-2 showed the presence of high mannose structures, (Man)n (GlcNAc)2, n = 9, 8, 7, 6. The S582A mutant was the most homogeneous with (Man)9(GlcNAc)2 comprising greater than 50% of oligosaccharides present. Analysis of purified VV WT and S582A mutant hCox-2 by liquid chromatography-electrospray ionization-mass spectrometry showed an envelope of peaks separated by approximately 160 Da, corresponding to differences of a single monosaccharide. The difference between the highest mass peaks of the two envelopes, of approximately 1500 Da, is consistent with the wild-type enzyme containing an additional high mannose oligosaccharide.