Albumin knockout (Alb ) mice exhibit a low plasma free fatty acid (FFA) concentration, but it was not known if the suppressed concentration reflects a lower rate of appearance (Ra) of FFA in the circulation (i.e., lower FFA flux) or if the absence of albumin alters the relationship between FFA flux and concentration. For understanding the role of albumin in FFA transport through the bloodstream, it is not sufficient to rely on FFA concentration data alone. Therefore, we developed a method to study FFA kinetics in Alb mice. Using an albumin-free formulation of U- Cpalmitate tracer, serum FFA kinetics were tested in Alb and wild-type (WT) mice. Results indicate that the flux of FFA in serum of Alb mice was significantly lower than in WT mice ( < 0.05), while albumin deficiency did not alter the relationship between FFA flux and concentration. Next, to test if suppressed lipolysis might have also been involved in the suppressed FFA kinetics, gene expression of a lipolytic enzyme (adipose triglyceride lipase, Atgl) and a marker of lipolysis (phosphorylation of hormone-sensitive lipase, p-HSL) were measured in adipose tissue. In contrast to the low FFA flux in Alb , both Atgl gene expression and p-HSL protein were significantly higher in adipose tissue of Alb than in WT mice ( < 0.05). Thus, the low FFA flux in Alb appeared to be driven by the absence of albumin's FFA binding functions rather than through regulation of lipolysis, indicating that albumin is an important factor in determining the flux of FFA in circulation. To improve understanding of the albumin protein's function in vivo, we tested plasma free fatty acid kinetics in albumin knockout mice compared with wild-type mice. Using a new tracer formulation strategy, it was discovered that the appearance rate of free fatty acids in serum is lower in albumin knockout mice than in wild-type mice. The results indicate that albumin is a major controller of free fatty acid kinetics.