The transgenic construct consisted of an 11.3 kb fragment of the human cholinergic gene locus, containing the first 5 exons of CHAT and the first coding exon of SLC18A3 (located in intron 1 of CHAT). A cassette containing cre followed by IRES-EGFP was inserted at the start codon of the SLC18A3 gene. EGFP expression was undetected. The 11.3 kb fragment included various promoter elements including a distal enhancer, RE1/NRSE, and NGFRE. Founder mice contained approximately 20 copies. Expression of cre recombinase by the human SLC18A3 promoter began at postnatal day 7 and was restricted to a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus. Maximal levels of expression were reached by 5 weeks of age,earlier onset than in the Tg(SLC18a3-cre)AMisa line. Transgenic expression was not observed in viscertomotor or other central or peripheral cholinergic neurons.