Aneutral trehalase gene (NTH1) was isolated from a kEMBL3 genomic library of the insect pathogenic fungus Metarhizium anisopliae. Sequencing of the gene revealed extensive homology with other fungal neutral trehalases. The NTH1 gene exists as a single copy in the genome. Two STREs exist in the 50UTR of NTH1, which may mediate transcriptional activation of the NTH1 gene in response to various stresses. The NTH1 gene encodes a protein of 737 amino acids with a calculated Mr of 83.1kDa. Acyclic adenosine 30,50-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site were found in the amino-terminal domain of NTH1, consistent with a regulatory enzyme. Expression of the trehalase cDNAwas achieved in Saccharomyces cerevisiae. Southern blot analysis of RT-PCR products indicated that the neutral trehalase gene is transcribed in vitro in cell-free haemolymph of the tobacco hornworm Manduca sexta and in vivo in the early stage of infection.