In past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous beta-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.