Single-strand-preferring nucleases (EC 3.1.30.1) selectively cleave internucleotide bonds in single-stranded regions of predominantly duplex DNA and DNA.RNA hybrids and extensively degrade denatured DNA and RNA. The functions of single-strand-preferring nuclease in plants are unknown. We have monitored this nuclease activity in flag leaves of wheat (Triticum aestivum L. cv. Chinese Spring) undergoing natural senescence and in primary leaves of wheat seedlings undergoing dark-induced senescence. In falg leaves, nuclease activity remained at basal levels during the first 2 weeks after anthesis, while chlorophyll content increased to a maximum. Nuclease activity then rose in concert with a decline in chlorophyll, reaching a 16-fold elevation at 5 weeks post-anthesis, when 53% of the chlorophyll had been lost. When 8-day-old wheat seedlings were induced to senesce by placing them in darkness, nuclease activity rose without apparent lag, reaching a 13-fold elevation in 7 days, when 61% of the chlorophyll had been lost. The increase in nuclease activity was reversible upon reexposure of seedlings to light, a decline beginning without apparent lag. Reversibility was complete for plants that had been held in darkness for 5 days, with activity returning to the control level in 2 days. These senescence-related changes in nuclease activity, measured in conventional assays, were consistent with concomitant analysis by activity staining of sodium dodecyl sulfate/polyacrylamide gels. We conclude that an increase in single-strand-preferring nuclease activity is closely associated with wheat leaf senescence and that nuclease activity is subject to negative photoregulation.