When cells and tissues of Drosophila are subjected to elevated temperatures, the pattern of protein synthesis shifts from the production of a broad spectrum of different proteins to the vigorous production of a small number of new, heat shock proteins. The intracellular distribution of these proteins has been investigated through autoradiographic analysis of cells labeled with 3H-leucine at 23 degrees and 37 degrees C. After examining sections of cultured cells from D. melanogaster and polytene cells of D. virilis by electron and light microscopy, we conclude that little (if any) heat shock protein becomes associated with mitochondria, despite the many lines of evidence linking the response to respiratory stress. Confirming earlier reports on the presence of heat shock proteins in nuclei, we find the proteins are very highly concentrated there and that their transport to the nucleus occurs very rapidly. Interestingly, their free concentration in the nuclear sap is extremely low; they are, in fact, quantitatively associated with chromosomes. This association occurs in a nonrandom manner, their concentration in highly condensed chromatin being very low relative to that of other chromosomal loci.