X chromosome inactivation results in the cis-limited inactivation of most, but not all, genes on one of the two X chromosomes in mammalian females. The molecular basis for inactivation is unknown. In order to examine the transcriptional activity of human X-linked genes, a series of mouse-human somatic cell hybrids under positive selection for the active or inactive human X chromosome has been created. Northern blot analysis of RNA from these hybrids showed that the human MIC2 gene, which is known to escape X inactivation, was transcribed in hybrids with either the active or inactive X chromosome. In contrast, the human TIMP gene was only transcribed in hybrids with an active human X chromosome. Further analysis using the polymerase chain reaction showed that there was at least one-hundred fold less transcription of the TIMP gene from the inactive X than from the active X chromosome. These findings demonstrate that the human TIMP gene is subject to X inactivation at the level of transcription, and illustrate the usefulness of the polymerase chain reaction to study the extent of X-linked gene repression by the process of X inactivation.