Previous studies indicate that the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca 2+ -dependent and attributed to the late Ca 2+ rise. However, its role in fertilization still remains unclear. Simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase occurred at the time of peak current (t p ) which corresponds to peak [Ca 2+ ] i , suggesting that NO is not related to any other ionic changes besides [Ca 2+ ] i . We measured O 2 consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. Our results suggested NO increased O 2 consumption. The fluorescence of reduced pyridine nucleotides, NAD(P)H was measured in controls and when the NO increase was eliminated by PTIO, a NO scavenger. Surprisingly, PTIO decreased the rate of the fluorescence change and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H 2 O 2 and caused weak and high fertilization envelope (FE). Our results suggest that NO increase upregulates NAD(P)H and H 2 O 2 production and consolidates FE hardening by H 2 O 2 .