The effect of metabolic or hormonal status on CoA biosynthesis was studied by comparing the rates of incorporation of [14C]-panthothenate into CoA in fasted and glucose-fed rats. Rat hearts and livers were freeze-clamped 1.5 hours after intravenous injection of [14C] pantothenate. CoA, pantothenate, and other pathway intermediates were separated by chromatography of tissue extracts on DEAE cellulose paper. Compared to the fasted rats, rats forced-fed glucose 0.5 hours before pantothenate injection 69% lower incorporation of radioactivity into CoA in liver, a 69% lower specific radioactivity of liver CoA, a 63% lower specific radioactivity of liver mitochondrial CoA, and a 44% lower incorporation of radioactivity in CoA in heart. The accumulation of labeled pathway intermediates was negligible. The cysteine content of liver was equal for the two conditions. There was no difference in level of unacylated CoASH for fasted and glucose-fed rats, suggesting that hormonal effects on degree of CoA acylation are not involved in this regulator mechanism. The specific radioactivities and concentrations of pantothenate in heart or liver were also nearly equal for the two conditions, so the regulatory mechanism does not involve hormonal effects on pantothenate uptake into tissues. Thus, the effect of glucose-feeding (and related changes of metabolite and insulin-glucagon levels) on the incorporation o[14C] pantothenate into CoA is exerted at a locus on the biosynthetic and/or degradative pathway between pantothenate and CoA.