The metabolism of the human hair follicle was investigated in vitro under conditions that maintained glycogen and adenosine triphosphate (ATP) content and the growth rate of the follicle at values observed in vivo. We have shown that only 10% of the total glucose utilized was oxidized to CO2 and 40% of this was oxidized via the pentose phosphate shunt. Although fatty acids and ketone bodies were oxidized by the hair follicle, they are poor energetic substitutes for glucose. Nor will fatty acids or ketone bodies sustain hair growth in vitro. Glutamine, however, was shown, both biochemically and by comparing growth rates, to be an important fuel with 23% of uptake being oxidized, generating a possible 2.16 +/- 0.32 nmoles ATP/follicle/h (mean +/- SEM) (glucose metabolism generates 4.54 +/- 0.61 nmoles ATP/follicle/h). Sixty-four percent of the glutamine taken up was calculated to be metabolized to lactate, showing that the hair follicle engages in both glycolysis and glutaminolysis. The glucose-fatty acid cycle appears to be unimportant in the hair follicle but our data indicates that a glucose-glutamine cycle does operate.