The PRimed IN Situ (PRINS) labeling method allows rapid, specific detection of human chromosomes in situ. We have adapted the PRINS protocol to mature human sperm in combination with a 3 M NaOH protocol for simultaneous in situ decondensation and denaturation of sperm nuclei. Using fluorochrome-labeled dNTPs in a sequential PRINS reaction, the direct detection of two or three distinct chromosomes must be performed within a timespan of 3 h. The method was tested with primers specific for chromosomes 8, 9, 12, 13, 16, 18, and 21 and the X. The frequencies of disomy ranged from 0.11 % to 0.34%. Chromosome-specific primers have been defined for most of the human chromosomes, including some that are indistinguishable by fluorescence in situ hybridization (FISH) with centromeric probes. Consequently, this new strategy constitutes a rapid and efficient alternative to FISH for detecting nondisjunction in human sperm.