Mice heterozygous for Robertsonian centric fusion chromosomal translocations frequently produce aneuploid sperm. In this study RBJ/Dn x C57BL/6J F1 males, heterozygous for four Robertsonian translocations (2N=36), were analyzed to determine effects on germ cells of error during meiosis. Analysis of sperm by three color fluorescence in situ hybridization revealed significantly elevated aneuploidy, thus validating Robertsonian heterozygous mice as a model for production of chromosomally abnormal gametes. Primary spermatocytes from heterozygous males exhibited abnormalities of chromosome pairing in meiotic prophase and metaphase. In spite of prophase abnormalities, the prophase/metaphase transition occurred. However, an increased frequency of cells with misaligned condensed chromosomes was observed. Cytological analysis of both young and adult heterozygous mice revealed increased apoptosis in spermatocytes during meiotic metaphase I. Metaphase spermatocytes with misaligned chromosomes accounted for a significant proportion of the apoptotic spermatocytes, suggesting that a checkpoint process identifies aberrant meioses. Immunofluorescence staining revealed that kinetochores of chromosomes that failed to align on the spindle stained more intensely for kinetochore antigens CENP-E and CENP-F than did aligned chromosomes. Taken together, these observations are consistent with detection of malattached chromosomes by a meiotic spindle checkpoint mechanism that monitors attachment and/or congression of homologous chromosome pairs. However, the relatively high frequency of gametic aneuploidy suggests that the checkpoint mechanism does not efficiently eliminate all germ cells with chromosomal abnormalities.