The distribution of N-linked glycans in rat testis has been probed using a panel of lectins derived from Galanthus nivalis (snowdrop, GNA), Canavalia ensiformis (jack bean, Con A), Lens culinaris (lentil, LCA), Pisum sativum (garden pea, PSA) and Phaseolus vulgaris, erythro- and leucoagglutinins (kidney bean, ePHA and lPHA). Several classes of N-linked glycan were identified in the spermatogenic series, and during differentiation into spermatozoa they altered in both their pattern of distribution and relative abundance. A population of tetra-antennary, non-bisected, complex glycans, detected by lPHA, was lost during the transition from spermatogonia to spermatocytes, while high-mannose structures were acquired; these were most abundant in spermatocytes, as were bi- and tri-antennary complex, non-bisected glycans, the latter becoming increasingly abundant on acrosomes and spermatozoa. Their bisected counterparts were more generally expressed throughout spermatogenic cells, although marked localization onto acrosomes and nuclear caps was again seen. Transition from spermatocytes to spermatids involved mainly changes of the acrosomal granule and nuclear cap, which were carried through to the final stages of differentiation. Sertoli cell surfaces and cytoplasmic granules showed a high level of N-glycan expression.