Simple sequence repeats (SSR), or microsatellites, are ubiquitous in eukaryotic genomes. Here we demonstrate the utility of microsatellite-directed DNA fingerprinting by polymerase chain reaction (PCR) amplification of the interrepeat region. No sequencing is required to design the oligonucleotide primers. We tested primers anchored at 3' or 5' termini of the (CA)n repeats, extended into the flanking sequence by 2 to 4 nucleotide residues [3'-anchored primers: (CA)8RG, (CA)8RY, and (CA)7RTCY; and 5'-anchored primers: BDB(CA)7C, DBDA(CA)7, VHVG(TG)7 and HVH(TG)7T]. Radioactively labeled amplification products were analyzed by electrophoresis, revealing information on multiple genomic loci in a single gel lane. Complex, species-specific patterns were obtained from a variety of eukaryotic taxa. Intraspecies polymorphisms were also observed and shown to segregate as Mendelian markers. Inter-SSR PCR provides a novel fingerprinting approach applicable for taxonomic and phylogenetic comparisons and as a mapping tool in a wide range of organisms. This application of (CA)n repeats may be extended to different microsatellites and other common dispersed elements.