The rapid induction of conserved heat shock proteins is mediated by the heat shock transcription factor HSF in response to various stressors. In the model organism Drosophila melanogaster evidence suggests that many heat-inducible genes remain to be identified. To accomplish this, we developed a cDNA microarray to measure changes in transcript levels. A comparison of the expression profiles of SL2 and Kc167 cells validated this tool. During a heat shock time course in SL2 cells, 131 genes showed altered expression. In contrast, studies in whole organisms revealed 687 heat-affected genes. Moreover, the response of larvae, adults, and cell lines to heat shock were distinct, and only slightly overlapped. Analysis of the temperature sensitive hsf4 mutant confirmed the HSF-dependence of most heat-regulated expression changes, save for the induction of hsp83 and CG8778. Further computational analysis enabled the formulation of a comprehensive view of the Drosophila heat shock response.