The parasitic protozoan Leishmania major differentiates in vitro, from the insect-adapted promastigote to the mammalian infective amastigote, in response to a temperature shift from 25 degrees C to 37 degrees C. We studied the genes encoding 70 kilodalton heat shock proteins (hsp 70 genes) in Leishmania substocks, which vary in their capability to differentiate. In total, four hsp 70 genes are arranged in tandem with intergenic regions of about 380 bp. These hsp 70 genes are 89% conserved at the aminoacid level when compared to the T. brucei hsp 70 genes. The expression of these four hsp 70 genes is increased, in vitro and in vivo, in response to a temperature shift from 25 degrees C to 37 degrees C. The parasite thus indeed responds to the transfer between hosts like it responds to a heat shock. In contrast, the high rate of transcription of a fifth identical hsp 70 gene, located at a separate locus, is unaffected by temperature shifts. The hsp 70 mRNAs have mini-exons trans-spliced onto their 5' ends and share unusually long (1000 nt) 3' untranslated extensions containing repetitive sequences. It is unclear whether or not the intergenic regions of the L. major hsp 70 genes function in transcription initiation and/or whether transcription results in the generation of polycistronic pre-mRNAs. Since each of the hsp 70 genes that we identified is expressed normally in an L. major substock that lost the capability to differentiate in response to an in vitro temperature shift, the inability to differentiate does not result from a general defect in the temperature-dependent control of transcription.