We have isolated a gene encoding a 70,000-Da heat shock protein (HSP70) from a chicken genomic library. The recombinant clone C411 was isolated by hybridization to a radioactively labeled plasmid containing Drosophila HSP70 gene sequences. The identity was established by hybrid selected translation using a subcloned restriction fragment, pC1.8, and total cytoplasmic RNA from chicken cells. The selected mRNA translated in vitro a product which co-migrates with in vivo synthesized chicken HSP70 by SDS-polyacrylamide gel electrophoresis. By Northern blot analysis of poly(A)+ mRNA from heat-shocked cells, we detected a 2.6-kilobase pair mRNA homologous to pC1.8. The nucleotide sequences within pC1.8 and a flanking 2.0-kilobase pair HindIII fragment contain a single contiguous open reading frame of 1905 nucleotides that corresponds to a protein of predicted size 70,000 daltons. Upstream of the initiator methionine codon, ATG, lie sequences homologous to the canonical transcription elements TATA, CCAAT, and SP1 binding site, and two overlapping heat shock elements. The order and spacing of these sequences share many features in common with the promoter for the human HSP70 gene.