Transcriptional stimulation of heat shock genes is generally due to the activation of heat shock transcription factor (HSF) 1. We demonstrate that in the murine leucemic cell line, P388, trimerization of HSF1, transcriptional activation of the hsp70 gene, and expression of Hsp70 are achieved as a result of heat shock. In contrast, the small heat shock proteins Hsp25 and alpha B-crystallin are not expressed in these cells and cannot be induced upon heat shock. Furthermore, no hsp25 transcript can be detected, indicating that there is a defect in the hsp25 gene or a block in its expression. Comparison of the hsp25 gene structure between P388 and Hsp25-expressing Ehrlich ascites tumor (EAT) cells by Southern blot analysis revealed no differences in the structural organization of the gene and no changes in its localization in the genome. However, sequence analysis of the hsp25 promoter region in P388 cells demonstrates minor differences. Despite these differences, the hsp25 promoter from P388 cells mediates heat shock-induced activation of a reporter gene when transfected into human HeLa cells which is comparable to that of the hsp25 promoter from EAT cells. Furthermore, the hsp25 gene isolated from EAT or P388 cells can both be expressed in HeLa cells and lead to a similar heat shock-stimulated accumulation of Hsp25. Silencing of the hsp25 and alpha B-crystallin genes in P388 cells by DNA-methylation could also be excluded since 5-azacytidine treatment does not influence expression of these genes. Interestingly, when expressed in P388 cells the hsp25 promoter from EAT cells is not activated upon heat shock, whereas the human hsp70 promoter is activated. Taken together, the data suggest cell line-specific differences in a mechanism of regulation of hsp25 transcription, which interferes with the activation of the promoter by HSF1 and which may also affect the alpha B-crystallin gene.