Proliferation and differentiation of mammary epithelia are regulated by the combined action of systemic hormones and locally derived paracrine growth factors. Keratinocyte growth factor (KGF) is a potential candidate stromal factor that may participate in the hormonal control of stromal/epithelial interactions. In this study, we have examined the in vivo effect of 17beta-estradiol (E) treatment on KGF expression in mammary glands of peripubertal (5-week-old) and mature (11-week-old) mice. Mice received subcutaneous injections of hormone after which KGF mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. E treatment caused a dose- and time-dependent increase in KGF mRNA levels in intact mice from both age groups. Neither 17alpha-estradiol nor progesterone injection affected KGF mRNA levels. Comparison of the relative expression of KGF in parenchyma-free fat pads and in intact glands demonstrated that the basal and E-dependent KGF mRNA levels did not require the presence of mammary epithelium. ELISA assay of KGF tissue content demonstrated that concomitantly with an up-regulation of mRNA, E treatment also increased KGF protein in mammary glands from peripubertal and mature mice. These data show that E treatment stimulates both KGF mRNA and protein expression in mammary stroma in vivo and raises the possibility that KGF has a role in E-regulated mammary gland development.