There are two functional insulin genes in the mouse genome. The Ins2 gene is imprinted and expressed monoallelically from the paternal allele in the yolk sac. In the present study we have re-examined the imprinting status of Ins1. We found that Ins1 is not expressed in the yolk sac of several laboratory mouse strains. The asynchrony of replication at the wild type locus was significantly lower than at imprinted loci and was more similar to non-imprinted loci. Finally, we have taken the advantage of the Ins1(neo) allele created by homologous recombination to examine the allelic usage at this locus. We observed that the neo gene inserted at the Ins1 locus was expressed from both the paternally and the maternally transmitted allele. Therefore, the Ins1 gene does not share any of the basic properties of imprinted genes. On the basis of these data, we concluded that Ins1 locus is unlikely to be imprinted in common laboratory mice.