Xiphophorus represents a valuable model for studying genomic contributions to neoplasia. For analyzing these contributions at the molecular level, basic information about the genome organization is a prerequisite. This study presents data on the organization and complexity of the genomes of three species of Xiphophorus, maculatus, variatus and helleri, representative of the problem. Their diploid nuclei, as measured in the erythrocyte, contain 1.19 pg, 1.23 pg, and 1.27 pg DNA, these values representing approximately 50% of that of birds, 20% of that of mammals. The melting curves of native, high molecular weight DNA are homogeneous, the Tm was determined for maculatus as 85.0° C (corresponding to a mean GC-content of 38.3%) for variatus as 86.0° C (GC=40.7%), for helleri as 85.0° C (GC=39.3%). Reassociation of sheared denatured DNA indicated approximately 90% single copy sequences, the remaining 10% are predominantly multiple copy sequences. The complexity of single copy DNA was determined from reassociation kinetics for maculatus as 3.97×108 base pairs, for variatus as 4.31×108 base pairs, and for helleri as 4.49×108 base pairs. The DNA of the three species upon isopycnic density gradient centrifugation in the presence of the fluorescence dye Hoechst 33258 shows in addition to the main band, two heavy (GC-rich) satellites, denoted in the order of increasing density, components I and II. Analytical centrifugation reveals for the main band DNA a buoyant density of 1.6980 gcm-3 (GC=38.7%), for component I 1.7080 gcm-3 (GC=48.9%), for component II 1.7150 gcm-3 (GC=56.1%). Each of the components comprises approximately 0.38% of the total DNA. Complete digestion of components I and II with restriction enzymes EcoRI and BamHI yields a complex banding pattern upon agarose gel-electrophoresis. A 2.4 kb fragment of component I and a 5.3 kb fragment of component II of helleri, cloned and amplified in the pBR322/E. coli RR1 system, hybridize efficiently to purified nuclei of liver. Furthermore, restriction fragments of component II DNA, transferred to nitrocellulose by Southern-blotting, hybridize with 18S and 28S ribosomal DNA.