Glutamine synthetase (GS) activity was measured in the caput and cauda regions of rat epididymis. Specific GS activity in the caput was 27-fold higher than that in the cauda. To compare GS activity within the epididymis to that within other tissues, specific and total GS activities were measured in the brain, liver, testes, kidney, and striated muscle. Caput epididymal specific GS activity was from 4- to 38-fold higher than GS activity in any other tissue; caput total GS activity was equal to that in brain. Epididymal GS activity was rapidly and completely inhibited by preincubation with methionine sulfoximine, a known inhibitor of GS. These results suggest that the high concentrations of GS activity in the caput epididymis may have functional significance in maintaining an optimal microenvironment for sperm maturation, perhaps by restoring luminal acid-base balance, removing ammonium and/or glutamate from the lumen, or supplying glutamine for the production of nucleic acids.