We have employed automated fluorescence-based technology to detect amplified tri-, tetra-, and pentanucleotide short tandem repeat (STR) loci electrophoresed on denaturing polyacrylamide sequencing gels. The system described incorporates an internal size standard in each sample, allowing the STR-PCR products to be sized automatically with a high degree of precision. By utilizing different fluorescent dye markers for loci that have overlapping allele size ranges, we have developed three multiplex STR systems containing a total of 14 different loci. These multiplex systems were then used to evaluate the usefulness of the 14 loci for the identification of individuals. Allele frequency data were collected from a minimum of 50 individuals from each of three different racial groups: Caucasians, Afro-Caribbeans, and Asians. Of the resulting 42 locus population sets, deviation from Hardy-Weinberg equilibria was detected in only the STR HUMCYARO3-Caucasian data. The probabilities of two unrelated individuals matching by chance (pM) at all 14 loci in the three multiplex reactions was < 1 x 10(14). The combination of multiplex STR-PCR and automatic fluorescence-based detection is thus a rapid and powerful technique for individual identification.