To identify previously undetected genes that may be involved in the transition from a resting state (G0) to a proliferative state (G1) of mammalian cells, we set out to isolate cDNA clones derived from mRNAs that appear in serum-stimulated cells in the absence of protein synthesis. A lambda cDNA library was prepared using poly(A)+ RNA from BALB/c 3T3 cells that had been brought to quiescence and subsequently stimulated with serum in the presence of cycloheximide. Approximately 50 000 recombinant phage plaques were screened, and 357 clones were isolated that hybridized to probes derived from stimulated-cell RNA but not to probes from resting-cell RNA. Cross hybridization analysis showed that four RNA sequence families account for approximately 90% of these clones. One of the clones hybridized to an actin probe; none hybridized to any of 13 oncogene probes tested. Five different RNAs that appear to be previously uncharacterized have been further analyzed. These RNAs accumulate and decay rapidly following stimulation by serum or purified growth factors, or by a tumor promoter, and they are superinduced by serum in the presence of cycloheximide. Three of the RNAs could be enriched by hybridization to cDNAs and translated in vitro, yielding proteins of approximately 43, 40 and 35 kd, respectively.