A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.