Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA Val(GUU) and a tRNA Val(GUA) gene. The method used involved a combination of classical D. discoideum parasexual genetics and molecular genetics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VII (A, H). Six tRNA Val(GUA) genes from the NC4 isolate were mapped: one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).