The high mobility group chromosomal proteins HMG-I and HMG-Y are closely related isoforms that are expressed at high levels in rapidly dividing, undifferentiated mammalian cells. We analyzed HMG-I/Y mRNA levels at various cell cycle stages in murine NIH/3T3 fibroblasts partially synchronized by seeding from quiescent, contact-inhibited cultures. Flow microfluorometric analysis of DNA content demonstrated a comparable degree of synchronization in such seeded NIH/3T3 cell populations as is obtained by serum deprivation or other means and has the added advantage of avoiding the use of possibly detrimental inhibitors or metabolic starvation to induce such synchrony. We show that HMG-I/Y mRNA levels gradually increase in NIH/3T3 cells during the first 16 h after seeding (G0/G1 to late S phase), but thereafter remain constant, in contrast to the cell cycle-regulated expression of the histone H3 gene. Although there is a 6-fold increase in HMG-I/Y expression during the transition from quiescent to proliferating NIH/3T3 cells, there is a much greater difference in expression (15- to 50-fold) among different cell types, possibly related to their state of differentiation. The HMG-I/Y mRNAs appear to be very stable; there was no decrease in their levels 6 h after actinomycin D transcription termination. The proportion of HMG-I to HMG-Y mRNAs was greater in the human than in the murine cells examined, appeared to be greater in proliferating than in quiescent cells, and did not always correspond with the HMG-I to HMG-Y protein ratio.