We have isolated and characterized genomic clones encoding a novel small heat shock (HS) protein (Hsp32) from Dictyostelium discoideum that is not homologous to the alpha-crystallin family Hsps. Besides its induction by HS, this gene is also regulated during the life cycle of the organism. At physiologic temperatures hsp32 is expressed at high levels in growing cells and at low levels in cells starved to initiate their developmental programme. However, in both cases the gene can be induced by HS. A DNA fragment containing the upstream region of hsp32 was shown to confer HS induction to a cat reporter gene, indicating a transcriptional regulation for this gene. A single transcription start site, located at position -152 relative to the initiator Met, 17 nucleotides downstream from a putative TATA box, was determined both in vegetative cells and cells starved for 6 h. This site was unchanged when either vegetative or starved cells were submitted to HS at 30 degrees C for 30 min. Despite HS induction, a perfect HSE element was not found in the 5' regulatory region of the gene. The hsp32 coding region is interrupted by a single intron located near its 5' end, which is properly spliced even under HS conditions.