After unilateral separation of the rat epididymis from the testis, the metabolism of various substrates in vitro by tissue from the attached and separated caput and cauda epididymidis at 7 and 28 days after surgery was determined by radiorespirometry. Hourly collections of 14CO2 were made during 5-hr incubations. The patterns of 14CO2 evolution from glucose indicated that most of the metabolic activity followed the Embden-Meyerhof glycolytic and the Krebs cycle respiration pathways. The alteration of the rate of glycolysis was always greater than that of respiration. In all samples, the metabolism of [2-14C]glucose was approximately equal to that of [6-14C]glucose (G-6) and less than that of [1-14C]glucose (G-1). Pentose cycle activity was indicated in all tissues from the caput and cauda epididymidis by the preferential utilization of G-1 over G-6. At 7 and 28 days after surgery, respectively, the G-1 :G-6 ratios of 14CO2 evolution after incubation for 2 hr were 9·75 and 7·79 for the separated caput, 5·17 and 2·66 for the intact caput, 3·11 and 2·52 for the separated cauda and 3·73 and 2·84 for the attached cauda epididymidis. Although epididymal separation did not affect the metabolism of [U-14C]glucose or [U-14C]fructose, glucose appeared to be a more important epididymal substrate than fructose.