Tyrphostins inhibit tyrosine kinases and have little effect on the activity of serine/threonine kinases. Pyruvate dehydrogenase kinase inactivates pyruvate dehydrogenase by phosphorylating serine residues within the multienzyme complex. This serine/theronine kinase represents a new family of protein kinases, and one (tyrphostin 47) of two tyrphostins tested appeared to activate the pyruvate dehydrogenase kinase as determined by [1-14C]-lactate oxidation to 14CO2. Experiments designed to determine if the tyrphostins altered pyruvate dehydrogenase activity in mitochondria prepared from rat epididymal adipocytes using [1-14C]-pyruvate as the substrate demonstrated a dose dependent increase in enzyme activity in the presence of tyrphostin 47, but not in tyrphostin 23. This apparent stimulation of pyruvate dehydrogenase activity was attributed to tyrphostin 47's ability to nonenzymatically decarboxylate [1-14C]-pyruvate, the substrate for the pyruvate dehydrogenase assay. Neither tyrphostin directly altered pyruvate dehydrogenase kinase activity. Therefore, assays utilizing [1-14C]-pyruvate and tyrphostin 47 are subject to analytical interference.