Alterations in the long-chain acyl-CoA binding to albumin in the carnitine palmitoyltransferase (CPT) assay appreciably affect the reaction at commonly used substrate concentrations. Since in the CPT assay the latter are typically well below saturation or V(max.) values, the measured enzyme activity depends on both the absolute quantity of albumin in the CPT assay and any biochemical modification of its binding. The present study verifies the striking dependence of the K0.5 for palmitoyl-CoA on albumin and the misleading 'activation' of the enzyme by compounds that also avidly bind to albumin. In assessing the intracellular physiological relevance of any modifier of CPT, the effects of protein binding in the assay assume particular importance. Indeed, any compound that alters CPT activity may do so, not directly, but as an assay artifact changing the free or unbound substrate concentrations.