The gene encoding the major heat shock-inducible member of the HSP70 family of Neurospora crassa was cloned and characterized. The 5 nontranscribed region shows the presence of consensus sequence motifs resembling the classical heat shock elements found in many heat shock-responsive eukaryotic promoters, as well as metal-responsive-element sequences. The coding region of the gene contains four introns with boundaries and internal consensus motifs typical of genes of filamentous fungi. None of the other stress-inducible hsp70 genes of fungal origin have, so far, been reported to contain introns. The sequence adjoining the transcriptional initiation zone shows the presence of prominent CT-rich stretches, characteristic of highly expressed fungal genes. The deduced amino acid sequence corresponds to a 646-residue polypeptide, with a calculated molecular mass of 70,561 Da and an average pI of 6.01, exhibiting strong sequence homology with many other eukaryotic HSP70s, with typical HSP70 family signatures 1 and 2 and a bipartite nuclear targeting sequence. Experiments with primer extension revealed the presence of one minor and two major transcriptional start sites. This gene, designated hsps-1, was mapped to a locus on the left arm of linkage group II, in close proximity to the AR-30 translocation breakpoint.The heat shock response is a fundamental attribute of living organisms, enabling them to withstand sudden environmental changes. The rapid synthesis of a specific complement of heat shock proteins (HSPs), or stress-responsive proteins, following hyperthermal injury or application of physiological or metabolic stress is recognized as an effective, protective strategy for ensuring cellular survival (32). In eukaryotic organisms, HSPs with a large molecular size (70 to 110 kDa) appear to be implicated in several functions critical for the maintenance of the integrity of structural proteins and cellular enzymes under unfavorable conditions.The stress-inducible HSP70 members compose one of the most highly conserved protein and gene families encountered throughout the biological world (4). The importance of the role of the stress-70 class of proteins as molecular chaperones of protein folding and oligomeric assembly, during heat shock as well as in normal cellular physiology, is beginning to be appreciated, and there is beginning to be a greater understanding of their structure-function relationships (13). The hsp70 multigene family of yeast, comprising eight members-including constitutive as well as stress-inducible ones-has been investigated intensively (6). In contrast, very little information is available on the heat shock genes and proteins of the filamentous fungi. Information derived from recent phylogenetic studies at the molecular level provides strong support for a close evolutionary linkage between the fungal and animal kingdoms (2). Therefore, elucidation of the evolutionary basis of defensive strategies elaborated by the filamentous fungi should provide invaluable genetic tools for exploration of similar ...