POU5F1has two alternatively spliced transcripts: a long (variant 1, NM_002701) and a short (variant 2, NM_203289) transcript. Only variant 1 is a key regulator of pluripotency. Hence, it is important to be able to distinguish this transcript from variant 2 and from the many pseudogenes present in the genome. Previous studies on the expression ofPOU5F1were, however, usually carried out without considering the existence of the two transcripts and the pseudogenes which could be the source of false positive RT-PCR amplification. Here, we establish an RT-PCR/restriction digestion analysis to distinguish variant 1 ofPOU5F1from variant 2 and all its currently known pseudogenes. Variant 1 hasApaI andTsp45I restriction sites, which are not present in the pseudogenes or in variant 2. Thus,ApaI- andTsp45I- digestions ofPOU5F1PCR fragment, amplified with primers flanking these sites, are sufficient to identify the true variant 1 ofPOU5F1. To study the expression of variant 2 ofPOU5F1, two forward primers in the 5′-region that are not present in variant 1 were combined with reverse primers located in exon 3 ofPOU5F1common to both transcripts. The assay was applied on 10 samples from peripheral blood leukocytes and commercially available ready-cDNAs from leukocytes and testis. We found that only variant 2 was expressed in leukocytes and testis and that the extracted RNA was not completely DNA free, despite DNAse treatment. This trace amount of DNA is a source of false positive RT-PCR amplifications. This article contains Supplementary Material available athttp://www.interscience.wiley.com/jpages/1045-2257/suppmat.