cDNA libraries have been generated from Nulli-SCCl murine embryonal carcinoma (EC) cells untreated or treated for 24 h with all-trans retinoic acid (RA) or hexamethylenebisacetamide (HMBA), two chemically unrelated inducers of differentiation of EC cells. The libraries were screened for gene sequences whose expression was differentially regulated by one or both compounds. Of 20,000 cDNA clones screened, only 12 showed reproducible quantitative differences. One of the latter clones (pH 34) has been studied in detail. pH 34 cDNA hybridizes with a polyadenylated RNA (650 nucleotides) which is abundant in untreated Nulli-SCCl EC cells but whose steady-state levels decrease within 6 h of exposure to HMBA, reaching a minimum at 24 h. RA has a less-marked effect on this mRNA. Addition of inducers to the cells in fresh medium produces an early (15 min) transient increase in pH 34 mRNA levels. Nuclear run-on experiments are consistent with the view that the decrease in pH 34 mRNA is due to post-transcriptional events. Subclones of pH 34 in pGEM-4 were used to synthesize mRNA which could be translated in vitro into a 14-kDa protein. DNA sequencing of the pH 34 cDNA revealed that it is 607 bp in length with a single open reading frame capable of encoding a protein of 118 amino acids. Primer extension experiments revealed that the insert contains the full 5' sequence. Comparison with known sequences failed to reveal significant homology with previously sequenced proteins.