The acyl-binding sites of avian fatty acid synthase have been further characterized using the fluorescent acetyl-CoA analog, beta-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl]alanyl coenzyme A (NBDA-CoA). Binding to nonsulfhydryl sites has been studied by treatment with neutralized hydroxylamine to remove NBDA bound in a thioester linkage. The binding of NBDA to hydroxylamine-insensitive sites can be divided into two types: "loose" and "tight." The NBDA bound at the loose site can be removed by rapid gel filtration through a centrifuge column or by added CoA and is kinetically competent to be the loading site for acyl groups. The NBDA binds to the tight site apparently irreversibly and inhibits the overall enzyme activity completely at a stoichiometry of approximately 2 per enzyme molecule. Pyridoxal 5'-phosphate can modify a large number of enzyme amino groups (greater than or equal to 20) and totally inhibits the enoyl reductase activity. This inhibition appears to be correlated with the binding of approximately four molecules of pyridoxal 5'-phosphate per enzyme molecule. Although the beta-ketoacyl reductase activity is unimpaired, the binding of NADPH at the sites involved in this activity is weakened. The fluorescence resonance energy transfer efficiency from NADPH, bound to pyridoxal 5'-phosphate modified enzyme, to enzyme-bound NBDA, or from NADPH, bound to unmodified enzyme, to NBDA bound only at the tight hydroxylamine-insensitive sites is not significantly different from the overall efficiency previously reported. This suggests that all of the acyl-binding sites are located approximately the same distance from the enoyl reductase and beta-ketoacyl reductase catalytic sites.