Brain acyl-CoA hydrolase (BACH) is responsible for most of the long-chain acyl-CoA hydrolyzing activity in the brain and is localized exclusively in neurons. There are two BACH isoforms: the major isoform, a 43-kDa BACH, and a lesser isoform, a 50-kDa BACH. In our previous work [Brain Res. Mol. Brain Res. 98 (2002) 81], a possibility was raised that these BACH isoforms might be generated from a single mRNA species via a mechanism of alternative use of translation start sites. However, the results obtained in the current study indicated that the 43-kDa BACH and 50-kDa BACH are not generated from a single mRNA species, but from distinct mRNA species transcribed by alternative use of transcription start sites. The molecular properties of the 50-kDa BACH were compared to those of the 43-kDa BACH. Palmitoyl-CoA hydrolase activity and protein stability were almost the same between both BACH isoforms. In addition, both 43-kDa BACH and 50-kDa BACH that were fused to green fluorescent protein showed cytosolic distribution. These results suggest that the 50-kDa BACH plays a similar role as the 43-kDa BACH. Therefore, since the 43-kDa BACH is expressed at higher levels than 50-kDa BACH, the 43-kDa BACH should largely contribute to understanding the physiological functions of the BACH gene in neurons.