The effect of dietary zinc deficiency on γ-glutamyl hydrolase (EC 220.127.116.11) activity and on pteroylpolyglutamate absorption was investigated in rats. Enzyme activity was determined in pancreas and gut lumen washings. Pteroylpolyglutamate absorption was studied by determining the rise in plasma folate levels following pteroylpolyglutamate ingestion. Two experiments were performed; in each purified diets were given to three groups of immature male Wistar rats for approximately 2 weeks. One group was given a Zn-deficient diet ad lib. (ZD), the second was pair-fed daily with this group on a Zn-adequate diet (PF) and the third was given the Zn-adequate diet ad lib. (AL). In Expt 1, significantly reduced pancreatic γ-glutamyl hydrolase activity was observed in ZD rats. In Expt 2, pteroylpolyglutamate was administered on day 14 and in the 3 h period following pteroylpolyglutamate ingestion, lumen γ-glutamyl hydrolase activity and plasma folate levels were significantly lower in ZD rats. Pancreas is reported as the source of lumen γ-glutamyl hydrolase in rats. The results presented indicate that the pancreatic enzyme is Zn-sensitive. It was concluded that, as a result, γ-glutamyl hydrolase activity was reduced in the lumen of ZD rats. Consequently the hydrolysis and subsequent absorption of pteroylpolyglutamate was impaired in ZD rats, as indicated by the smaller rise in plasma folate levels that occurred following pteroylpolyglutamate ingestion. Results of this study concur with previous observations in human beings and rats that Zn deficiency has an adverse effect on folate metabolism.