The purpose of the present study was to examine whether impaired milk folate secretion during maternal iron deficiency is due to an altered flux of folates within the mammary secretory cell. Specifically we sought to determine whether the folate substrates of methionine synthase and the products of folylpolyglutamate synthetase are altered during iron deficiency in vivo. Rats were fed diets containing 0.5, 2.0 or 7.0 mg/kg folate and 8(Fe-) or 250(Fe+)mg/kg Fe throughout gestation and lactation. On day 17 of lactation dams were milked and killed. The concentration of reduced, methylated (5-CH3-H4), nonmethylated short and long chain forms of folate in milk were determined using a differential microbiological technique. Total mean milk folate concentrations among Fe- dams fed 2.0 and 7.0 mg/kg folate were half that of Fe+ dams fed 2.0 and 7.0 mg/kg folate. Despite this, the relative proportion of reduced, 5-CH3-H4, short and long chain folates did not differ in milk from Fe+ or Fe- dams. Approximately 75% of milk folates were methylated. Only Fe+ dams fed 7.0 mg/kg folate produced milk containing significant quantities of incompletely reduced folates. In conclusion, activity of the mammary epithelial cell enzymes methionine synthase and folylpolyglutamate synthetase in vivo, are unaffected by iron deficiency and therefore are not responsible for the dramatic reduction in milk folate secretion.