Technical Abstract: Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report (ATVB 11:1944, 2009) suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for conjugating fatty acids. This report cast doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA conjugation induced phosphorylation of JNK and ERK, and TLR target gene expression in THP1 monocytes or RAW264.7 macropages, respectively when cultured in a low FBS (0.25%) medium. C12:0 induced NFkB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Since BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike suspension cells (THP-1), BSA conjugation is required for C16:0 to induce TLR target gene expression in adherent cells (RAW264.7). BSA conjugated C16:0 transactivated TLR2 dimerized with TLR1 or TLR6, and through TLR4 as with C12:0. These results and additional studies with LPS sequester Polymixin B and MyD88-/- macrophages indicated that that SFA-induced activation of TLR2 or TLR4 is a fatty acid-specific effect, but not due to contaminants in BSA or fatty acid preparations.