To better understand how infections by mycoplasmas affect gene expression in human cells, we quantitatively measured the transcripts of 38 cytokine genes in HPV E6- and E7-immortalized cervical and prostatic epithelial cells before and after infection by four human urogenital mycoplasmas, M. fermentans, M. genitalium, M. hominis and M. penetrans. Using the multi-probe RNase protection assay (RPA), 22 and 23 cytokine gene transcripts were detected in the non-infected control prostatic and cervical epithelial cells, respectively. Although there were no discernible changes in cell morphology and growth kinetics following 72 h of mycoplasmal infection, 55-74% of the cytokine genes expressed in the two human epithelial cell lines were altered. Most changes reflected an increased expression of these cytokine genes, while expression of some cytokine genes significantly decreased. The effects varied with host cell type and species of infecting mycoplasmas. These alterations in gene expression were more profound in the cervical epithelial cells than in the prostatic cells. M. fermentans produced the most significant effects, followed by M. penetrans, M. genitalium and M. hominis. Some alterations in the gene expression were transient, but most persisted over the course of chronic (9 months) mycoplasmal infection. Prolonged gene expression changes induced by chronic mycoplasmal infection may gradually alter important biological properties in the infected mammalian cells and produce a unique form of disease process.