BACKGROUND: Accurate gender determination is crucial in many scientific disciplines, but especially so in prenatal diagnosis of X-linked diseases and forensic investigations. Today, molecular techniques, especially typing for a length variation in the X-Y homologous amelogenin gene (AMELX and AMELY), are used for gender assignation. This amelogenin test is an integral part of most PCR multiplex kits that are used for DNA profiling, but in 1998 there was a report of two normal males being typed as female with this test. Subsequently, a small number of amelogenin negative (or AMELY null) males have been reported in various populations but little data are available characterising these deletions. AIMS: The study aims to determine the size of the deletion in five AMELY null males by typing DNA samples for markers surrounding this gender-determining locus. The possible relationships among the AMELY null samples are examined through analysis of their deletion size and associated Y-chromosome microsatellite haplotypes. We also attempt to determine the frequency of AMELY negative males in Australia. SUBJECTS AND METHODS: DNA samples from five AMELY null males, from different geographical regions, were made available for this study. The samples were typed for eight sites, all located on the short arm of the Y chromosome, using PCR and gel electrophoresis. Eleven Y-chromosome specific microsatellites were also typed on each sample in order to generate haplotypes for phylogenetic analysis. A questionnaire was sent to all Australian forensic centres requesting information on the frequency of AMELY negative males observed in their laboratories. RESULTS: Two different sized deletions were seen in the five AMELY null samples. One deletion (in two samples) has a size of between 304 and 731 kbp, whereas the other (in three samples) ranges between 712 and 1001 kbp. Y-microsatellite haplotypes indicate that the smaller deletion is probably identical in the two samples, but this is not the case with the larger deletion. The frequency of AMELY negative is rare in Australia, with an overall frequency of 0.02%. CONCLUSION: Comparisons of both deletion size and haplotypes with published data suggest that most AMELY nulls are the result of independent evolutionary events, even in those populations where the frequency is relatively high. Although AMELY null males are extremely rare in most populations, typing an additional gender-determining locus should be considered in forensic investigations where the reference sample is of unknown gender.