Difficulties encountered in the cloning of DNA from Streptococcus pneumoniae and other AT-rich organisms into ColE1-type Escherichia coli vectors have been proposed to be due to the presence of a large number of strong promoter-acting sequences in the donor DNA. The use of transcription terminators has been advocated as a means of reducing instability resulting from disruption of plasmid replication caused by strong promoters. However, neither the existence of promoter-acting sequences of sufficient strength and number to explain the reported cloning difficulties nor their role as a source of instability has been proven. As a direct test of the ''strong promoter'' hypothesis, we cloned random fragments from S. pneumoniae into an E. coli vector containing transcription terminators, identified strong promoter-acting sequences, and subsequently removed the transcription terminators. We observed that terminator removal resulted in reduced copy numbers for the strongest promoter-acting sequences but not in reduced promoter strengths or altered plasmid stabilities. Our results indicate that promoters strong enough to require transcription terminators for plasmid stability are probably rare in S. pneumoniae DNA.