The mouse prion protein (PrP) gene (Prn-p), which encodes the only macromolecule that has been identified in scrapie prions, is tightly linked or identical to a gene (Prn-i) that controls the duration of the scrapie incubation period in mice. Constellations of restriction fragment length polymorphisms distinguish haplotypes a to f of Prn-p. The Prn-pb allele encodes a PrP that differs in sequence from those encoded by the other haplotypes and, in inbred mouse strains, correlates with long scrapie incubation time (Westaway et al., Cell 51: 651-662, 1987). In segregating crosses of mice, we identified rare individuals with a divergent scrapie incubation time phenotype and Prn-p genotype, but progeny testing to demonstrate meiotic recombination was not possible because scrapie is a lethal disease. Crosses involving the a, d, and e haplotypes demonstrated that genes unlinked to Prn-p could modulate scrapie incubation time and that there were only two alleles of Prn-i among the mouse strains tested. All inbred strains of mice that had the Prnb haplotype were probably direct descendants of the I/LnJ progenitors. We established the linkage relationship between the prion gene complex (Prn) and other chromosome 2 genes; the gene order, proximal to distal, is B2m-II-1a-Prn-Itp-A. Recombination suppression in the B2m-Prn-p interval occurred during the crosses involved in transferring the I/LnJ Prnb complex into a C57BL/6J background. Transmission ratio distortion by Prna/Prnb heterozygous males was also observed in the same crosses. These phenomena, together with the founder effect, would favor apparent linkage disequilibrium between Prn-p and Prn-i. Therefore, transmission genetics may underestimate the number of genes in Prn.