The isoprenoid pathway leads to various essential non-sterol products in insects. These end products have a crucial role in growth, differentiation, sexual maturation, and reproduction. 3-Hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase (EC 4.1.3.5.) has generally been considered one of the committed steps of the pathway. We had previously reported the cloning of a cytosolic HMG-CoA synthase cDNA in Blattella germanica; we have now isolated and characterized a new cDNA clone for HMG-CoA synthase in this insect. Analysis of this 1716-base pair cDNA reveals a deduced protein of 455 residues with a molecular mass of 51,424 Da. The two HMG-CoA synthases have 69% identical amino acid residues, and both lack an N-terminal leader peptide to target the protein into mitochondria. This HMG-CoA synthase cDNA can revert the Chinese hamster ovary-K1-derived cell line, Mev-1, which is a defective mutant for HMG-CoA synthase. Both HMG-CoA synthase genes are expressed differently throughout development. Analysis of adult tissues shows higher expression in ovary and fat body. The expression of HMG-CoA synthase (EC 4.1.3.5.) and reductase (EC 1.1.1.34) genes during the gonadotrophic cycle in B. germanica shows that the three genes of the isoprenoid pathway are developmentally regulated in the ovary.