The function of seven promoters from Corynebacterium glutamicum, P-hom, P-leuA, P-per, P-aes1, P-aes2, P-45, and P-104, was analyzed in a heterologous background. DNA fragments carrying the promoters were cloned into shuttle promoter-probe vectors replicating in Escherichia coli and C. glutamicum (pET2), Streptomyces lividans (pGL7011) and Bacillus subtilis (pRB394). With the exception of P-hom, P-leuA and P-104 in B. subtilis, all promoters were found to be active in all species. Non-radioactive methods of primer-extension analysis and of S1-nuclease protection assay using automatic sequencer were developed to determine the respective transcriptional start points (TSPs). All TSPs were determined by primer extension and in two promoters (P-45 and P-hom) the main TSPs were confirmed by S1-mapping. While the main TSPs were identical in all four species, utilization of multiple TSPs varied among the species and additional TSPs were detected in S. lividans. Knowledge of the efficiency of promoters and of exact respective TSPs may be of practical value for the construction of expression systems in a heterologous background.