The identification of signals that regulate pluripotentiality and self-renewal is fundamental to the understanding of stem cell biology. To quantify the functionally active genome of pluripotent R1 embryonic stem (ES) cells, we used the method of serial analysis of gene expression (SAGE) to sequence a total of 140,313 SAGE tags. Of 44,569 unique transcripts, 9% matched known genes in the nonredundant GenBank database, whereas >35% of the unique tags did not match any known mouse sequence. Comparisons of relatively abundant (> or = 20) tags in the ES cell SAGE catalog with publicly available SAGE data sets identified 16 transcripts with an abundance profile unique to pluripotent R1 ES cells. We confirmed 12 by RT-PCR including those encoding KLF2, a transcription factor; galanin, a hypothalamic neurohormone; BAX, a proapoptotic signaling factor; and CDK4 and PAL31, cell cycle progression associated proteins. The data from this study provide a starting point for detailed transcriptome analyses of stem cells.