Although adrenal glucocorticoids were known to be important for adequate milk production, little is known about their effects on the metabolism of mammary glands during lactation. In this study, lactating Wistar rats on the 12th day of lactation were divided in the following groups: sham-operated (SO) and adrenalectomized (ADX) receiving no treatment; SO and ADX starved for 24 h and refed intragastrically with 2.5 ml of 50% glucose solution, 2 h before the experiment (SOR and ADXR) and ADX receiving substitute therapy with dexamethasone (ADX + DEX). Sacrifices were performed 2 days after surgery. Weight, lipid content and in vivo lipogenesis rate were evaluated in mammary gland (M.GLAND), liver, parametrial white adipose tissue (PARA) and interescapular brown adipose tissue (BAT). ATP citrate lyase activity was measured in M.GLAND, liver and PARA of SO, ADX and ADX + DEX. The rate of lipogenesis and 14CO2 production from 14C-glucose by isolated acini from M.GLAND and plasma glucose were also determined. In ADX rats, food intake, lipid content, in vivo lipogenesis rate and ATP citrate lyase activity in M.GLAND were significantly lower than those in SO rats. The M.GLAND lipogenesis rate of SOR group was similar to the value found in SO rats. In ADXR rats, the M.GLAND lipogenesis rate was not normalized. However, the therapy with DEX elevated lipid content, in vivo lipogenesis rate and ATP citrate lyase activity to levels similar to those in SO. These results suggest that the glucocorticoids are essential for the occurrence of normal lipid synthesis in M.GLAND during lactation.