Collagen V is a quantitatively minor component of collagen I fibrils and the defective product of classic Ehlers-Danlos syndrome (EDS). To provide new insights into its embryonic function, a continuous evaluation of the expression pattern of proalpha1(V), a chain common to all collagen V molecular forms, was performed by in situ hybridization of developing mouse from 7.5 days after conception (dpc) to birth. Proalpha1(V) transcripts were first detected at 8.5 dpc, signals being considerably augmented at 16.5 dpc and declining at birth. Hybridization signals were, at first, exclusively detected in the dorsal aorta wall, heart, and adnexa. At 10.5 dpc, col5a1 expression was found in the heart, dorsal aorta wall, branchial arches, mesonephrotic tubules, and intestinal mesenchyme and coincided with proalpha1(I) developmental expression. Later stages exhibited an intense signal in more restricted regions, notably the skin, the bones and vertebral column, the cornea, the tendons and ligaments, the peritoneal membranes, the umbilical cord, and the salivary gland. The data revealed the important contribution of collagen V to the development of functional connective tissues. Proalpha1(V) signals were exclusively detected in the flattened cells of the surface ectoderm at 10.5 dpc. By 12.5 dpc, when cells had become cuboidal, the signal switched to the dermal fibroblasts. Thus, type V collagen appears to contribute to epidermis differentiation. Our data also suggest that collagen V participates in bone formation and/or mineralization and in the renewal of stromal cells in the cornea. The results underscore the role of collagen V in developing embryos and provide important clues for analyzing the phenotype of mouse models for EDS.