Larvae of two cold-hardy insects, a freeze tolerant Eurosta solidaginis and a freeze avoiding Epiblema scudderiana species, were sampled at different times over a winter season and the activities of three mitochondrial enzymes were measured. Citrate synthase (CS), glutamate dehydrogenase (GDH), and NAD-isocitrate dehydrogenase (NAD-IDH) activities all decreased by 50% over the mid-winter compared with autumn values. In Eurosta solidaginis all activities returned to initial values by mid-April, but decreased again in late April when the larvae pupated. In Epiblema scudderiana, activities of GDH and NAD-IDH increased in the spring, but CS activity remained relatively unchanged from mid-winter on. To determine the influence of temperature alone on enzyme activities, mid-October larvae acclimated to -4 or 15 degrees C were compared; results showed greater activity of NAD-IDH at 15 degrees C than at -4 degrees C in Eurosta solidaginis (P < 0.05 by the Student's t-test), but a lower activity of GDH in Epiblema scudderiana at 15 degrees C (P < 0.05). Other enzyme activities were unchanged by acclimation. Reduced activities of mitochondrial enzymes in mid-winter are consistent with winter dormancy of these species, and could reflect lower mitochondrial numbers at this time. The regulation of these seasonal changes appears to be largely a response to cues other than short-term temperature acclimation,