By two-dimensional (2-D) genome typing, i.e., electrophoretic separation of restriction enzyme-digested genomic DNA on the basis of both size and sequence in denaturing gradient gels followed by hybridization analysis, several hundred alleles (spots) can be analyzed in parallel, using a micro- or minisatellite core probe. We studied the segregation of 213 and 214 spots detected by microsatellite core probe (CAC)n and minisatellite core probe 33.6, respectively, in two three-generation human pedigrees. Reproducibility of the spot patterns was such that particular spot variants could be scored in both pedigrees. Between 73 and 74% of the spots scored were variant and were transmitted in a Mendelian manner. Very little cosegregation among the 2-D spots themselves was observed, suggesting a random distribution over the genome. Several pairs of spots that appeared to contain both alleles from single loci were identified. The few spots detected by both probes (overlapping spots) showed different segregation patterns, indicating that each probe detects independent sets of genetically informative loci. These results provide a firm basis for using 2-D DNA typing to identify disease loci and for constructing a 2-D spot genetic linkage map of the human genome.