Interleukin-10 (IL-10) has been shown to attenuate lipopolysaccharide (LPS) stimulation of inducible nitric oxide synthase (iNOS) in various cell types. Guanosine triphosphate cyclohydrolase I (GTPCH) and type-2 cationic amino acid transporter (CAT-2) are enzymes that regulate iNOS activity. We therefore sought to assess the effects of IL-10 on the expression of these regulatory enzymes in LPS-stimulated macrophages that are known to express iNOS. Five minutes after adding LPS to these macrophage cultures, various doses of recombinant human IL-10 were also added. The samples were harvested for analysis 18 h after exposure to both LPS and IL-10. In LPS-stimulated macrophages, IL-10 attenuated the upregulation of nitric oxide and iNOS protein but not iNOS mRNA. IL-10 also attenuated the LPS-induced upregulation of CAT-2 mRNA. However, IL-10 and LPS had no effect on GTPCH mRNA expression. We therefore conclude that IL-10 inhibits nitric oxide formation in LPS-stimulated macrophages partly by decreasing iNOS protein expression. Moreover, our data suggests that transcriptional control of CAT-2 plays a role in IL-10 mediated influences upon nitric oxide biosynthesis.