In situ hybridization at high resolution with biotin-labeled DNA was used to locate specific transcriptional units within the chromosomal puffs of normal heat shock loci and of new heat shock loci generated by transformation. This method resolves copies of the hsp70 gene that are separated by 40 kb within the 87C heat shock locus. In the case of two new puff loci generated by transformation, the heat-activated transcript is encoded by sequences that reside entirely within the puff domain and the activated promoter is positioned asymmetrically within the puff. However, an adjacent promoter and its transcriptional unit which do not appear to be induced by heat shock are also within the puff. These results support the idea that a chromosomal puff is a structure that facilitates or results from vigorous transcription, but that the structural change alone is insufficient to induce transcription.