Respiration rate, time to pupation and the expression patterns of selected genes were examined during the diapause to post-diapause transition in the alfalfa leafcutting bee, Megachile rotundata held at constant 4 degrees C in winter storage. Respiration quotients were at or below 0.7 from December to May and then increased to over 0.8 in June and July. The time required for prepupae to reach the pupal stage following transfer to 29 degrees C decreased from 23 days in December to 10 days in July. HSP70 was expressed at a consistently high level in all the diapausing prepupae stored at 4 degrees C. In contrast, we demonstrated previously that HSP70 expression in diapausing prepupae maintained under field conditions began decreasing after December and was expressed at trace levels in the June samples. Transferring prepupae stored at 4 to 25 degrees C at monthly intervals from December to July induced a significant decrease in HSP70. Levels of HSC70 showed no changes during the transition to post-diapause development in prepupae maintained at 4 degrees C. Transferring the prepupae to 25 degrees C during the April-June time interval elicited an increase in HSC70 expression. HSP90 was expressed at a consistent level in prepupae stored at 4 degrees C but decreased to very low levels after being transferred to 25 degrees C in December-February prepupae: no decrease was noted in the April-July prepupae. Actin was expressed at trace levels in the diapausing prepupae maintained at 4 degrees C and increased slightly in the post-diapausing pupae. Transferring prepupae stored at 4 to 25 degrees C at monthly intervals from December to July induced an increase in actin expression. These results indicate that the level of gene expression for selected genes in diapausing and post-diapause bees is highly influenced by their thermal history.