Collagen requires hydroxylation of its proline residues to achieve proper assembly, structure, and function. Prolyl 4-hydroxylase catalyzes formation of 4-hydroxyproline, which is essential for collagen triple helix formation and stability. Prolyl 3-hydroxylase catalyzes formation of 3-hydroxyproline, which is far less abundant in collagens and whose function remains unclear. Recently mutations in prolyl 3-hydroxylase 1 have been associated with osteogenesis imperfecta, yet the temporal and spatial expression patterns of the prolyl 3-hydroxylase family members during development and in adult tissues remain undefined. By northern blot analysis distinct differences in transcript sizes of the three prolyl 3-hydroxylase genes were detected. Quantitative RTPCR demonstrated tissue-specific differences in prolyl 3-hydroxylase expression, most notable of which were high levels of prolyl 3-hydroxylase 2 in kidney and prolyl 3-hydroxylase 1 expression in embryonic tissues. Finally, in situ hybridization was used to assess spatio-temporal distribution of three prolyl 3-hydroxylases at embryonic days 11–15. Importantly, prolyl 3-hydroxylase 1 was expressed within cartilage condensations of the vertebral bodies and in the aortic arch of the developing heart, whereas prolyl 3-hydroxylase 2 was expressed in developing lens capsule. The prolyl 3-hydroxylase 3 gene showed more generalized expression overlapping somewhat with the other two genes. This report characterizes expression of the three prolyl 3-hydroxylase genes in embryonic and adult mice. Overall these data demonstrate tissue specific prolyl 3-hydroxylase gene expression in both fetal and adult tissues indicating a developmental role for prolyl 3-hydroxylase activity.