Background Platelet-activating factor (PAF) is a potent signaling phospholipid produced by embryos to facilitate standard embryonic development and subsequent implantation. Along with PAF, intracellular calcium is required for the initiation and facilitation of developmental transitions in embryos. Previous literature has revealed that PAF and intracellular calcium are interconnected during early embryo development; however, the signaling mechanism is unknown. This study reports enhanced induction of intracellular calcium release in response to exogenous PAF administration in sea urchin embryos at the two-cell stage. Methods Gravid Lytechinus variegatus sea urchins were injected with 0.5M KCl to induce release of gametes. In a 100 µL drop of seawater, 20 µL of eggs and 2 µL of sperm were combined. Once the inseminated eggs developed a fertilization membrane, a 5 µM concentration of a fluorescent calcium indicator (Fluo-4) was added and maintained in culture. Embryos were subsequently cultured to the two-cell stage over the next 90 minutes. Following incubation, 45 µL of two cell embryos were transferred to a poly-L-lysine-coated microscope slide, after which PAF (10-7M) was added. This addition of PAF was performed while the slide was imaged under a fluorescent microscope (Echo Revolve) to observe the fluorescence of calcium release, which was recorded as relative fluorescence units (RFU). The procedure was repeated using the biologically inactive form of PAF (Lyso-PAF) as a negative control. Data was analyzed via paired t test (Sigma Plot). Results A total of 16 two-cell stage embryos were utilized as described. PAF (mean= 70.9 RFU) induced a significant (P< 0.01) rise in intracellular calcium levels over background (controls; mean= 44.5 RFU). Lyso-PAF was not observed to induce any rise in intracellular calcium levels over background levels. Conclusion These results indicate that there is increased intracellular calcium release in embryos that have been exposed to exogenous PAF in comparison to control embryos and embryos that have received Lyso-PAF. Due to the known separate actions of PAF and intracellular calcium during development combined with our findings, this demonstrates that PAF likely induces the release of intracellular calcium, facilitating the development of sea urchin embryos.