We have detected the presence of a small (2.95 kb) plasmid in a clinical isolate of Streptococcus pneumoniae. A restriction map was constructed for this plasmid and for pDP1 (the only previously reported pneumococcal plasmid); no apparent differences were observed and the two plasmids hybridized strongly to each other. Portions of pDP1 were then cloned into Escherichia coli K-12, using the vector pUC19, and the pneumococcal DNA insert was used as a probe to screen 500 clinical isolates of S. pneumoniae for pDP1 sequences. The plasmid was detected in a total of 8 isolates. These were of various serotypes and no correlation could be found between the presence of the plasmid and the geographical location from which it came, the type of infection, or with resistence to antibacterial drugs. Although no function has yet been assigned to pDP1, it may form the basis of a useful vector for cloning in S. pneumoniae, as it contains at least seven unique restriction sites.