The diversity and steady-state concentration of single copy gene transcripts in the exponentially growing HeLa cell have been measured by determining the nucleotide sequence complexities of heterogeneous nuclear RNA, total polyribosomal messenger RNA and poly(A) +mRNA. The reassociation kinetics of HeLa cell DNA fragments 320 nucleotides in length reveal that a minimum of 62% of the HeLa cell genome consists of non-repetitive sequences having a C 0t 1 2 † † Abbreviations used: C 0 t, the product of nucleic acid concentration (in moles of nueleotides per liter) time (in seconds); C 0t 1 2 , the value of C 0 t at which 50% of a measured reassociation or hybridization reaction is complete; hnRNA, heterogeneous nuclear RNA; hnRNP, heterogeneous nuclear ribonucleoprotein; PIPES, piperazine-N, N′bis(α-ethane sulfonic acid). of 2238 m · s, corresponding to a complexity of 1.25 × 10 9 nucleotide pairs. A non-repetitive DNA fraction was isolated from 3H-labeled HeLa cell DNA by three successive cycles of kinetic fractionation and was determined to be at least 97% pure non-repetitive sequences as indicated by the kinetics of its reassociation with total HeLa DNA. In RNA-excess measurements, HeLa cell hnRNA saturated at least 13.3% of the non-repetitive DNA tracer in a kinetically heterogeneous reaction that extended over at least three decades of RNA C 0 t and which did not reach termination even at the highest RNA C 0 t measured (2.2 × 10 5 m · s). In contrast, polyribosomal mRNA saturated 1.49% and poly(A) +mRNA saturated 1.33% of the tracer in reactions which terminated and displayed singlecomponent pseudo-first-order kinetics. Based on the fraction of the tracer in hybrid at the highest RNA C 0 t value the sequence complexity of HeLa cell hnRNA is estimated to be, at minimum, 4.1 × 10 8 nucleotides, representing one-third of the double-stranded information in the HeLa cell genome. The kinetics of hybridization of hnRNA demonstrated the existence of at least two abundance classes. One of these, the “intermediate” hnRNA class, has a complexity of 1.22 × 10 8 nucleotides and consists of sequences present at an average abundance of 0.4 copy per cell. The other is a “rare” class of hnRNA which has a complexity of 2.59 × 10 8 nucleotides, corresponding to 63% of the total measured hnRNA complexity. Sequences belonging to this rare hnRNA class are estimated to be present at an average frequency of only 0.02 copies per cell, or one sequence in every 50 cells. Total polyribosomal mRNA has a complexity of 4.59 × 10 7 nucleotides and is estimated to be present at an average abundance of four copies of each sequence per cell. Taking the average size of HeLa cell messenger RNA to be 1750 nucleotides, the measured complexity of total polyribosomal mRNA corresponds to a repertoire of 26,200 different mRNAs. Poly(A) +mRNA has a complexity of 4.1 × 1.0 7 nucleotides and each sequence is estimated to have an average abundance of one copy per cell. The fact that the sequence complexity of total polyribosomal mRNA is slightly higher than that of poly (A) +mRNA suggests the possibility that a set of as many as 2800 mRNA species may be restricted to the fraction of HeLa cell mRNA that does not bind to oligo(dT)-cellulose, and which may therefore lack poly (A). The present results are discussed in relation to previous complexity measurements, and some of their biological implications are considered. Of particular significance is the unexpected observation that 93% of the complexity of HeLa cell hnRNA is represented by sequences present at considerably less than one copy per cell, including many that are estimated to possibly reflect the transcription of some single copy DNA sequences only once per cell cycle.