Initial velocity, product, dead-end, and substrate inhibition studies are described for gentamicin acetyltransferase I in the forward reaction. Initial velocity patterns were linear with tobramycin (Km acetyl-CoA = 1.7 micron, Km tobramycin = 1.6 micron), concave upward with sisomicin (V/K.Et = 1.7 X 10(7) M-1 s-1, indicative of nonrapid equilibrium conditions), and showed partial uncompetitive substrate inhibition with gentamicin C1a (Km acetyl-CoA = 1.3 micron, Km gentamicin C1a = 0.12 micron, KI = 6.5 micron). Product inhibition by CoA and acetyltobramycin consists of two competitive and two noncompetitive patterns. Dead-end inhibition by butyryl-CoA and neomycin consists of two competitive and two uncompetitive patterns. However, the uncompetitive pattern between neomycin and acetyl-CoA became noncompetitive when sisomicin rather than tobramycin was the nonvaried substrate. These results are consistent with a Random BiBi mechanism with synergism between binding sites which is nonrapid equilibrium with gentamicin C1a and sisomicin and rapid equilibrium with poor substrates such as tobramycin. The substrate inhibition by gentamicin C1a arises from a reduction in the rate of product (CoA) release which is partially rate-determining under nonrapid equilibrium conditions.