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The cholinergic mouse neuroblastoma cell line NS20Y was adapted to undifferentiated growth in suspension culture. When suspension cells were transferred to surface culture and treated with dibutyryl cyclic AMP, the cells underwent differentiation as assessed by biochemical, morphological, and physiological criteria. Differentiated NS20Y cells in co-culture with mouse muscle cells had the capacity to form functional neuromuscular junctions with the muscle cells. The sequence complexities of the poly(A)-containing messenger RNA (poly(A)+ mRNA) of the differentiated, process-forming cells (P-cells) and undifferentiated cells in suspension culture (S-cells) were measured by analysis of the kinetics of hybridization of the mRNAs with their complementary DNAs (cDNAs). There were less than 100 high abundance and approximately 8000 low abundance poly(A)+ mRNAs in both differentiation states. Heterologous hybridization reactions and recycling of the cDNA probes revealed that 9.7% and 6.8% of the messages in P- and S-cells, respectively, were specific to those differentiation states. The P-cell-specific sequences included approximately 3 high abundance and 320 low abundance poly(A)+ mRNAs. The S-cell-specific sequences included approximately 3 high abundance and 250 low abundance poly(A)+ mRNAs. We conclude that the increment in NS20Y differentiation results in both the disappearance of old, and the appearance of new mRNAs in polyribosomes.