Prdm1 mRNA was detectable by RT-PCR in all cell cultures in which Prdm1 immunostaining was found and in all tissue regions from which cells were obtained for culture; incubation with the C-terminal peptide used for immunization blocked immunostaining by the corresponding antibody ; on immunoblots and in cultures, staining was eliminated in controls that either lacked the Prdm1 antibody, had non-immune serum instead of the Prdm1 antibody, or had a nonPrdm1 antibody; and Prdm1 immunostaining was almost completely eliminated by treating somite-containing explants with antisense RNAi oligonucleotides that were designed to specifically target Prdm1 mRNA, whereas staining was not affected by treatment with a scrambled RNAi that was not specific to Prdm1. In addition to our results with somitic myocytes, we found that Prdm1 was also expressed in Anidulafungin myoblasts obtained from Azoramide embryonic limbs, as well as in the differentiated fast and fast/slow myocytes and myotubes that were formed from these embryonic myoblasts. Two main types of embryonic myoblasts are obtained from E4 limbs: 60�C70% of the myoblasts are committed to form small myocytes or myotubes that express only fast MyHC and the remaining 30�C40% of the myoblasts are committed to form myotubes that co-express both fast and slow MyHCs. We confirmed the formation of these fast and fast/slow types of myotubes in E4 cultures, and we found that Prdm1 was expressed in the nuclei of both types of myotubes. Because none of the three slow MyHC isoforms is expressed by fast myotubes in E4 cultures, this result showed that Prdm1 expression was not limited to differentiated cells that expressed slow MyHC, but was also found in myotubes that expressed only fast MyHC. In addition, we found that Prdm1 was expressed in 80�C90% of the Pax7-positive myoblasts in the E4 cultures. This result suggests that Prdm1 was expressed by both types of myoblasts in E4 cultures, the,60�C70% of the total myoblasts which were of the fast type and the remainder which were of the fast/slow type.Finally, we found that Prdm1 was also expressed in fetal E12 myoblasts and the myotubes they formed in culture. Fetal myoblasts are distinct from and replace embryonic myoblasts as development proceeds.