1. We investigated the effects of serum albumin on inducible nitric oxide synthase (iNOS) expression in RAW 267.4 macrophages. Crude fraction-V type albumin as well as bovine serum albumin filtrated for endotoxin induced concentration-dependent iNOS expression in macrophages. Accordingly, NO production (estimated by supernatant nitrite) was markedly (up to 10-fold) increased in the presence of albumin. 2. Albumin-induced expression of iNOS protein was inhibited by cycloheximide and NO production was abolished after incubation of the cells with an iNOS inhibitor, N(G)-monomethyl-l-arginine (LNMMA). 3. An inhibitor of the NF-kappaB pathway, pyrrolidine dithiocarbamate (PDTC), as well as inhibitors of JAK2/STAT and ERK, AG490 and U0126, respectively, significantly reduced albumin-induced iNOS expression and NO production, while an inhibitor of the p38 pathway, SB203580, did not significantly affect NO production induced by albumin. 4. Both types of serum albumin were contaminated with traces of endotoxin. The endotoxin levels were found not to be sufficient for the observed induction of nitrite production in RAW 267.4 cells. In addition, the albumin-stimulated induction of iNOS was not reduced by preincubation of albumin-containing media with polymyxin B, a LPS inhibitor. 5. Polymerised albumin fractions were detected in the commercially available albumin tested in this study. A monomeric albumin-rich fraction, separated by ultrafiltration, showed a potent inducing effect on iNOS expression and NO production, while a polymer-rich fraction showed a smaller effect. 6. Advanced glycation endproducts (AGE) of albumin were not formed by interaction with glucose in incubation medium, as AGE was not increased even after long-time (4 weeks) incubation in albumin-containing media [3.2-4.4 microg ml(-1) (basal) vs 4.8-5.6 microg ml(-1) (in glucose-containing media)]. However, the duration of albumin exposure to glucose influenced the basal stimulatory properties of albumin. 7. Our results suggest that serum albumin fractions, as gained by cold alcoholic extraction, may include determinants that stimulate or further enhance stimulation of RAW 267.4 cells and are different from endotoxin, polymeric albumin and AGE.