hsp30 is a small heat shock protein of Neurospora crassa which earlier studies suggested may associate with mitochondria during cellular heat shock. We show here that the association of hsp30 with mitochondria is reversible and that hsp30 dissociates after cells are returned to normal temperature. We sequenced the gene for hsp30 and defined its transcript by S1 nuclease analysis and cDNA sequencing. The gene apparently is present in the genome as a single copy, and it contains no introns. The encoded 25.3-kDa peptide is related to other small heat shock proteins, especially those from green plants. According to its deduced sequence, hsp30 can form two strongly amphiphilic alpha-helices, including one at its amino terminus. In binding assays, in vitro synthesized hsp30 bound strongly to mitochondria isolated from heat-shocked cells but not to mitochondria prepared from cells incubated at normal temperature. A mutant hsp30 peptide, deleted in the amino-terminal amphiphilic helix, bound more avidly than the full-length hsp30 to mitochondria isolated from heat-shocked cells and exhibited less stringent requirements for binding. The mutant peptide also showed strong affinity for mitochondria isolated from unstressed cells.