Most molecular work on insect diapause has focused on the expression of unique diapause transcripts, rather than the protein products. Here we present our first results from a proteomic comparison of diapausing and nondiapausing pupal brains. Proteins extracted from diapausing pupal brains of the flesh fly Sarcophaga crassipalpis were separated by two-dimensional gel electrophoresis and compared with those from nondiapausing pupal brains. Unique proteins and proteins expressed at different levels in diapausing and nondiapausing brains were identified by Nano-LC/MS/MS (capillary-liquid chromatography-nanospray tandem mass spectrometry). With this approach and Coomassie staining we detected 17 unique or upregulated (&ge3x) spots, and 11 spots that were downregulated in diapause. Heat shock proteins (HSP70 and several small HSPs) were among the most conspicuous brain proteins present in higher amounts during diapause. Brain proteins that were less abundant in diapause included phosphoenolpyruvate synthase, fatty acid binding protein, EG0003.7, and an endonuclease. Our 2-D proteome maps included several additional unknown proteins that are more abundant in either the diapause or nondiapause brains. While the mRNAs encoding certain of these proteins (e.g.HSPs) were previously known to be associated with diapause, the other proteins were not known to be linked to diapause, thus suggesting that the proteomic approach nicely supplements work done at the transcript level.