The mechanisms that promote and regulate transcription in mycoplasmas are poorly understood. Here, a promoter-probe vector based on the pMTnTetM438 minitransposon and containing a promoterless lacZ reporter gene was constructed to analyse Mycoplasma genitalium transcription in vivo. Recovered transposon insertions were in monocopy, with 16 % expressing enough beta-galactosidase (beta-Gal) to yield colonies exhibiting a detectable blue colour. A sample of 52 blue colonies was propagated and selected for further analyses. The beta-Gal activity of the corresponding cultures was measured to quantify, in a reproducible way, the transcription levels of the interrupted ORFs. Several insertions were found in sense with the interrupted ORF, but surprisingly there was also a number of insertions in non-coding regions, many of them in repetitive DNA regions known as MgPa islands. Moreover, 30 % of the analysed transposon insertions had the lacZ gene in the opposite orientation to the coding frame, suggesting the existence of antisense transcripts that may be involved in the control of gene expression in M. genitalium.