Although drug-resistance markers have been used frequently for gene-disruption mutagenesis in Streptococcus pneumoniae, none has yet been shown to be free of dependence on local transcription for its expression. Indeed, the erythromycin-resistance marker (erm), originating in pAM beta 1, has been used as an indicator of local transcription on several occasions. A procedure is demonstrated for evaluation of the autonomous expression of such a marker by placing it in a consistent background, at the pneumococcal ami (aminopterin resistance) locus, in combination with active or inactive alleles of the ami promotor (pA). Using this test platform, a chloramphenicol-resistance marker (cat) and a spectinomycin-resistance marker used in streptococcal gene disruption studies and derived from pJS3 and pDL269, respectively, were shown to depend on local transcriptional signals for expression when placed in the pneumococcal chromosome as single-copy genes. To overcome this limitation, new drug-resistance cassettes were designed and constructed, using pA as a model for synthetic promoters for the erm and cat genes. Both new cassettes were shown, by the same procedure, to be expressed after insertion in the pneumococcal chromosome, independent of local transcription. A new insertion-duplication vector, pEVP3, incorporating the new cat cassette and a lacZ reporter derived from pTV32, was also constructed. The ami test platform was used to demonstrate both the autonomous expression of cat and the reporter function of lacZ in chromosomal copies of pEVP3.