Population estimates of the Devils Hole pupfish (Cyprinodon diabolis) extending over a 25-year period reveal an annual population cycle that reaches a low during spring and a high during fall. The four earliest years (1972–1976) preceded a U.S. Supreme Court water rights decision intended to protect pupfish habitat. Mean and maximum population sizes during those four years were significantly lower than during subsequent periods examined through 1997, and annual minimum population size was significantly lower during 1972–1976 than during 1991–1997. Annual maximum population size is positively correlated with water level for the month in which it occurred as well as for the month one year prior to the time of the estimate. The lowest historic water levels occurred during the period 1972–1976, and over the past 60,000 years comparable or lower water levels may have occurred only during the altithermal, about 6000 years before the present. During these two periods the Devils Hole pupfish population has probably experienced its most severe bottlenecks.