The R-spondin (Rspo) proteins constitute a novel class of ligands that induce Wnt signalling. Rspo1 knockout XX mice were previously shown to be sex-reversed, but some remain sub-fertile. These last were unable to feed their pups for some unknown reason. Using these mice and transplanted mammary tissues from Rspo1(-/-) virgin mice in nude mice, we report that the lack of Rspo I expression results in the absence of duct side-branching development and subsequent alveolar formation, explaining the above mentioned phenotype. Our data demonstrate that local epithelial Rspo1 signalling is required for normal development of the mammary gland. (C) 2009 Elsevier Inc. All rights reserved.