The kidney plays a major role in carnitine biosynthesis, excretion and acylation. Unlike in the rat, human kidney contains all enzymes needed to form carnitine from trimethyllysine in activities exceeding those of the liver. This carnitine precursor is found to be increased in plasma of patients with chronic renal failure. Free carnitine formed in the kidney as well as carnitine reabsorbed from the glomerular filtrate may be acylated in the proximal tubule. Isolated rat cortical tubule suspensions contain total carnitine concentrations of 2.85 mumols/g protein. During incubation over 60 min the acylcarnitine/carnitine ratio decreased, indicating deacylation of acylcarnitine in proximal tubules. Exogenous carnitine was acylated at a rate of 35 mumols/h.g protein. Besides pyruvate and acetate, ketone bodies stimulated the acylation rate severalfold, indicating that these substrates are a major source of acetyl-CoA for the acylation reaction. This may explain the higher acetylcarnitine/carnitine ratio found in urine under ketotic conditions.