An overview of gene cloning in Streptococcus pneumoniae is presented. The advantages of such cloning, especially for pneumococcal genes, are enumerated. The molecular fate of DNA in transformation of S. pneumoniae, in particular, the conversion of DNA to single-strand segments on entry, determines the mechanisms for plasmid establishment and interaction with the chromosome. One of these mechanisms, the chromosomal facilitation of plasmid establishment, is useful for obtaining recombinant plasmids and for introducing an allele from the chromosome into a plasmid. The difference between linear and circular synapsis of donor DNA strands with the chromosome is illustrated. Circular synapsis can give rise to circular integration, which is useful for insertional mutagenesis of chromosomal genes, for coupled cloning in Escherichia coli, and for sequential cloning of DNA along the pneumococcal chromosome. Cloning in S. pneumoniae is not notably affected by DNA mismatch repair or restriction systems in the host cell. Unusual features of gene expression in S. pneumoniae are discussed. Transcription begins most often at promoters with extended -10 sequences, and in a small but significant number of cases, translation does not require a ribosome-binding site with a Shine-Dalgarno sequence.