Spot 14 (S14) is a 17 kD protein located in tissues that synthesize fatty acids. In the lactating mammary glands of S14 null mice there is significantly reduced triglyceride content due to a 70% reduction in lipogenesis with a 90% reduction in medium-chain fatty acids. However, the site and role of the S14 protein in the regulation of lipid synthesis remains unknown. We observed no difference in gene expression or in vitro activity of all tested lipogenic enzymes in wild-type vs null mice. A sensitive recycling assay measuring mammary gland malonyl-CoA revealed that malonyl-CoA is significantly increased in S14 null mice vs wild type (7.19 ± 0.68 vs 5.44 ± 0.43 nmol/g, p < .05). Therefore, the block in lipogenesis occurs at the terminal step, fatty acid synthase (FAS). Based on this evidence, we hypothesized that S14 is integral to the function of FAS, which catalyzes the production of fatty acids from malonyl-CoA. We first tested whether S14 acts by binding fatty acids during their production or transport. Recombinant Spot 14 produced from baculovirus-infected insect cells was used to investigate whether S14 acts as a fatty acid binding protein (FABP). We utilized a well-established assay that binds a hydrophobic fluorescent probe, 1-anilinonaphthalene 8-sulfonic acid (ANS), to putative FABPs. We found that the S14 protein bound to ANS with positive cooperativity (Hill coefficient = 2.9), suggesting that S14 is a multimer, and the formation of the multimer creates the hydrophobic pocket for ANS binding. However, the fluorescent probe was not displaced by fatty acids or their CoA derivatives of varying lengths. Therefore, to investigate further which specific lipogenic proteins associate with S14 in vitro, we created an affinity agarose column to which we bound the purified S14. Elution at 3M NaCl followed by SDS-PAGE showed only a single protein of molecular weight greater than 200 kD. This high-affinity interaction is consistent with S14 binding to FAS. Our data suggest that S14 possesses a hydrophobic binding pocket and interacts with FAS in vivo and in vitro to regulate the rate of fatty acid synthesis in mammary tissue.