ABSTRACT: The split ejaculation technique concentrates the most motile and viable spermatozoa in the first part of the ejaculate. Several clinicians use this fraction for insemination and in vitro fertilization. Eleven vasovasostomized men with antisperm antibodies participated in this study. The mixed antiglobulin reaction (MAR) for IgG, IgA, and secretory component was carried out on the whole ejaculate and the fractions of the split ejaculate. The isotype and concentration of free antisperm antibodies were determined in serum, the whole ejaculate and the fractions of the split ejaculate by the indirect MAR and the tray agglutination test. The detection of bound antisperm antibodies revealed almost identical reactions in fractions one and two and in the whole ejaculate. The results suggested that the majority of antisperm antibodies in the ejaculate from vasovasostomized men are transuded from serum not only at the epididymal and/or the prostatic level but also in the seminal vesicles.