The X-inactive-specific transcript (Xist) locus is a cis-acting switch that regulates X chromosome inactivation in mammals. Over recent years an important goal has been to understand how Xist is regulated at the initiation of X inactivation. Here we report the analysis of a series of targeted mutations at the 5' end of the Xist locus. A number of these mutations were found to cause preferential inactivation, to varying degrees, of the X chromosome bearing the targeted allele in XX heterozygotes. This phenotype is similar to that seen with mutations that ablate Tsix, an antisense RNA initiated 3' of Xist. Interestingly, each of the 5' mutations causing nonrandom X inactivation was found to exhibit ectopic sense transcription in embryonic stem (ES) cells. The level of ectopic transcription was seen to correlate with the degree of X inactivation skewing. Conversely, targeted mutations which did not affect randomness of X inactivation also did not exhibit ectopic sense transcription. These results indicate that X chromosome choice is determined by the balance of Xist sense and antisense transcription prior to the onset of random X inactivation.