The contribution of the POU domain, class 5, transcription factor-1 (POU5F1) in maintaining totipotency in human embryonic stem cells (hESCs) has been repeatedly proven. In humans, two isoforms are encoded: POU5F1_iA and POU5F1_iB. So far, no discrimination has been made between the isoforms in POU5F1 studies, and it is unknown which isoform contributes to the undifferentiated phenotype. Using immunocytochemistry, expression of POU5F1_iA and POU5F1_iB was examined in hESCs and all stages of human preimplantation development to look for differences in expression, biological activity, and relation to totipotency. POU5F1_iA and POU5F1_iB displayed different temporal and spatial expression patterns. During human preimplantation development, a significant POU5F1_iA expression was seen in all nuclei of compacted embryos and blastocysts and a clear POU5F1_iB expression was detected from the four-cell stage onwards in the cytoplasm of all cells. The cytoplasmic localization might imply no or other biological functions beyond transcription activation for POU5F1_iB. The stemness properties of POU5F1 can be assigned to POU5F1_iA because hESCs expressed POU5F1_iA but not POU5F1_iB. However, POU5F1_iA is not the appropriate marker to identify totipotent cells, because POU5F1_iA was also expressed in the nontotipotent trophectoderm and was not expressed in zygotes and early cleavage stage embryos, which are assumed to be totipotent. The expression pattern of POU5F1_iA may suggest that POU5F1_iA alone cannot sustain totipotency and that coexpression with other stemness factors might be the key to totipotency.