The sequential arrangement of nucleosomes along the chromatin fiber is punctuated by highly nuclease-sensitive sites. We previously mapped such sites to the 5' terminus of several heat shock genes in Drosophila by a novel indirect endlabeling technique. Such preferentially accessible sites in chromatin may function as points of entry to the DNA for RNA polymerase and control proteins. We have developed an exonuclease protection technique for mapping protein binding sites in chromatin, and have found two such sites for both the hsp 82 and hsp 70 genes. Site I is present before and after heat shock gene activation, and covers the TATA box sequence, whilst site II surrounds the upstream heat shock control element and appears only during heat shock. We suggest that heat shock genes are activated by the sequential binding of at least two protein factors, and we have developed new chromatin-binding and DNA-binding procedures to detect these proteins in crude nuclear extracts. We are now using these procedures as assays for the purification of the two proteins using standard column chromatography and high performance of the two proteins using standard column chromatography and high performance liquid chromatography.