We obtained a unique CD46 cDNA, STC/CY4, from the human testis, the predicted amino acid sequence of which suggested the presence of a novel isoform of CD46. This message was present predominantly in the testis, and the predicted isoform possessed a short (11 amino acids) transmembrane section (TM) and an unidentified cytoplasmic tail (CY). When expressed in Chinese hamster ovary (CHO) cells, this CD46 isoform underwent no O-glycosylation and was mostly retained in the endoplasmic reticulum. This unusual behaviour of the new isoform was due in part to the short TM and the unusual sequences of the CY. The molecular mass of this isoform was 42000, approximately 20000 smaller than conventional CD46. These properties of the STC/CY4 isoform were similar to those of sperm CD46. The only difference between sperm CD46 and the STC/CY4 isoform expressed on CHO cells was that only the latter possessed N-linked sugars of high mannose types. Since the STC/CY4 isoform may behave like sperm CD46 in cellular localization and posttranslational modification, studies of sperm-egg interassociation were performed using hamster eggs and CHO cell clones expressing various isoforms including the STC/CY4. Rosette formation was seen most effectively between hamster eggs and STC/CY4-expressing CHO cells. These results infer that O-glycosylation perturbs CD46-mediated sperm-binding to eggs and thus sperm CD46 lacking O-linked sugars can serve as an adhesion molecule. The possible role of CD46 in fertilization and the structural differences between sperm and conventional CD46 are discussed.