We have partially purified the messenger RNA coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 220.127.116.11) from HeLa cells and obtained a cDNA clone containing part of its sequence. Using this clone to probe electrophoregrams of RNA transferred to nitrocellulose, we have investigated the characteristics of nuclear and cytoplasmic transcripts in these cells. In the cytoplasm, nature GAPDH mRNA was detected in Northern blots as an intense band, apparently unique, of approximately 1400 nucleotides. The half-life of this mRNA was determined both from the decay kinetics, after a chase with actinomycin D, and from the labeling kinetics during an accumulation experiment. Both kinds of experiments yielded a half-life value of about 8 h, while the accumulation experiment indicated that steady-state GAPDH mRNA amounted to about 1.6% of cytoplasmic poly(A)-rich RNA. Much longer species, likely to be restricted to the nucleus, were also detected in RNA extracted from total cells. At least three discrete species of 1600, 4000, 5800 and 6800 bases were observed above a trailing background extending up to about 8000 bases. This value is commensurate with a functional size of the GAPDH transcription unit in the order of 13000 bases, which we determined by measuring the size of the ultraviolet inactivation target. Until direct evidence can be obtained at the genomic level, the present results provide the first clue to the existence of introns, presumably at least four, in a GAPDH gene from a higher eucaryote.