The spontaneously occurring mutation in op rats produces an animal with severe osteopetrosis. The op mutation has been localized to a 0.51 cM portion of rat chromosome 10. Lying within the genetic critical region for op is the Tbl3 (transducin beta‐like protein 3) gene. The Tbl3 protein resembles the transducin superfamily of proteins that are known to regulate cellular functions such as cell division, cell‐fate determination, gene transcription, transmembrane signalling, mRNA modification and vesicle fusion. In this study, we tested if this gene could be responsible for the osteopetrosis seen in the op rat. M‐13 tailed primer sets were designed from rat genomic sequence to cover the 22 exons of the rat Tbl3 gene. These primer sets were amplified by PCR against genomic DNA from affected mutant animals and the two parental strains, BN and LEW. Mutational analysis of the Tbl3 sequences from the three strains failed to reveal significant differences between affected mutant animals and the two parental strains although single nucleotide polymorphisms (SNPs) between BN and the other two strains were identified. These data indicate that the osteopetrosis seen in the op rat is not the result of a mutation within the coding region of the Tbl3 gene. Analysis of additional functional and positional candidate genes will be required to identify the causative mutation in this animal model of failed bone homeostasis.