A 11.2-kb fragment containing the ectABC genes of the biosynthetic pathway of ectoine from the Gram-positive, moderately halophilic bacterium Halobacillus dabanensis D-8(T) was obtained by inverse polymerase chain reaction. Subsequently, the entire ectABC cluster was cloned and analyzed. It revealed that the intergenic regions of the ectABC genes from H. dabanensis D-8(T) are more tightly spaced than those of Chromohalobacter salexigens, Halomonas elongata, Marinococcus halophilus, and Salibacillus pasteurii. The amino-acid sequence deduced from ectABC was highly homologous that from Virgibacillus pantethenticus (EctA 52%, EctB 60%, EctC 67%, respectively). The ectABC genes were cloned in the expression plasmid pMXB10 resulting in pMXB10ectABC. The ectoine was detected from cell extract in Escherishia coli ER2566 containing pMXB10ectABC using C-13 nuclear magnetic resonance spectroscopy.