Rat mammary gland proteomes at day 21 (prepubertal) and day 50 (late puberty) were compared by 2D difference gel electrophoresis. Two-hundred fifty-one spots were significantly different ( p < 0.05) in abundance. Peptide mass fingerprint analysis of a subset of these proteins identified two significantly over-represented classes including structural and blood proteins (increased), and metabolism-relevant proteins (reduced) in day 50 relative to day 21 glands. This is a first report of mammary gland proteome differences at these important breast cancer-relevant time-points.