The genetic control of susceptibility to many common diseases, including cancer, is multigenic both in humans and in animals. This genetic complexity has presented a major obstacle in mapping the relevant genes. As a consequence, most geneticists and molecular biologists presently focus on "single gene" diseases. To make the multigenic diseases accessible to genetic and molecular analysis, we developed a novel genetic tool, the recombinant congenic strains (RCS) in the mouse (4). The RC strains are produced by inbreeding of mice of the second backcross generation between two inbred strains, one of which serves as the "donor" and the other as the "background" strain. A series of RCS consists of approximately 20 strains, each carrying a different set of genes: approximately 12.5% genes from the common donor inbred strain, the remaining 87.5% from the common background inbred strain. As the set of donor strain genes in each RC strain is different, the nonlinked genes of the donor strain involved in the control of a multigenic trait, e.g., cancer susceptibility, become distributed into different RC strains where they can be analyzed one by one. Hence, the RCS system transforms a multigenic trait into a series of single gene traits, where each gene contributing to the multigenic control can be mapped and studied separately. Recently we demonstrated that the RCS system is indeed capable of resolving multigenic traits, which are hardly analyzable otherwise, by mapping four new colon tumor susceptibility loci (8; P. C. Groot, C. J. A. Moen, W. Dietrich, L. F. M. van Zutphen, E. S. Lander, and P. Demant, unpublished results). For successful application of the RCS system, extensive genetic characterization of the individual recombinant congenic strains is essential. In this paper we present detailed information about the genetic composition of three series of RC strains on the basis of typing of 120-180 markers distributed along all autosomes. The data indicate that the relative representation of the donor strain genes in the RC strains does not deviate from the theoretical expectation, and that the RC strains achieved a very high degree of genetic homogeneity and for all practical purposes can be considered inbred strains. The density and distribution of markers reported here permits an effective mapping of unknown genes of donor strain origin at almost all autosomal locations. Much of this information has been obtained using the new class of genetic markers, the simple sequence repeat polymorphisms.(ABSTRACT TRUNCATED AT 400 WORDS)