Background and Aim: Concentrations in blood of plasminogen activator inhibitor type 1 (PAI-1) and circulating free (non-esterified) fatty acids (FFA) are increased in diabetes and may accelerate atherosclerosis. We have shown that FFA increase expression of PAI-1 by activation of a transcription factor that binds to the repeated sequence 5′-TG(G/C) 1-2CTG-3′. This study was designed to determine whether FFA chain length, saturation, or both affect agonist properties and whether agonist properties are mediated by activated, thioesterified FFA (fatty acyl-CoA). Methods and Results: Human hepatoma cells were exposed to selected FFA associated with bovine serum albumin (BSA). Triacsin C (5 μM) was used to inhibit production of fatty acyl-CoA. PAI-1 was assayed by enzyme linked immunosorbent assay. Maximal induction of PAI-1 was similar with medium and long chain FFA (fold induction of PAI-1 accumulated in conditioned media compared with control: C10=1.8±0.1, C12=2.0±0.1, C14=2.0±0.2, C16=1.4±0.1, C18=1.6±0.1, C20=1.32±0.1, p<0.005 for each compared with control). Increased unsaturation did not alter the agonist properties of FFA (fold induction with C16:0=1.4±0.1, C16:1=1.4±0.1; C18: 0=1.6±0.1, C18:1=1.5±0.1, C18:2=1.6±0.1, C18:3=1.4±0.1 and C20: 4=1.3±0.1, C20: 5=1.4±0.1, n=6). However, maximal effects were seen with lower concentrations of longer chain FFA. Triacsin C consistently attenuated effects of FFA. Conclusions: Longer chain FFA exhibit maximal effects at lower concentrations. Augmented expression of PAI-1 is mediated by the fatty acyl-CoA derivative. These criteria identify targets for therapy designed to normalize expression of PAI-1 and retard progression of atherosclerosis in subjects with elevated concentrations of FFA in blood including those with insulin resistance.