The globular domain in the NH2-terminal propeptide (N-propeptide) of the proa1(I) chain is encoded by exon 2 of the Col1a1 gene and has been implicated in a number of processes that are involved in the biogenesis, maturation, and function of type I collagen. These include intracellular chain association, transcellular transport and secretion, proteolytic processing of the precursor, feedback regulation of synthesis, and control of fibrillogenesis. However, none of these proposed functions has been firmly established. To evaluate the function of this procollagen domain we have used a targeted mutagenesis procedure to generate mice that lack exon 2 in the Col1a1 gene. Mouse lines were established on both a mixed 129 Hsd/Sv and C57Bl/6 background and a pure 129 OlaHsd/Sv background. Adult mice on the mixed background are normal in appearance and are fertile. To the extent that they have been studied, procollagen synthesis, secretion, and proteolytic processing are normal in these mice, and collagen fibrillogenesis is only slightly altered. However, breeding of heterozygous mutant mice on the 129 background generated homozygous mutants at less than the expected frequency. These findings suggest that while the N-propeptide is not essential for collagen biogenesis in mice it may play some essential role during embryonic development.