The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.