We have isolated a maize ubiquitin cDNA clone which encodes one partial and three full-length, identical 76 amino acid repeats, in a polyprotein conformation. The deduced amino acid sequence of the mature monomeric polypeptide is identical to that determined for three other plants, barley, oat, and Arabidopsis, and differs from yeast and animal ubiquitin by only two and three amino acids, respectively. Hybridization of the cDNA clone to restriction endonuclease-digested genomic DNA revealed that ubiquitin is encoded by a small multigene family in maize. Northern blot analysis of poly(A)(+) RNA indicated that multiple ubiquitin mRNAs of 2.1, 1.6 and 0.8 kb are produced in maize shoots and roots. The abundance of the largest (2.1 kb) of these transcripts increased transiently 3- to 4-fold over the first 1 to 3 h in seedlings that were subjected to heat shock, and then returned dramatically within 1 h almost to the preshocked level. In contrast, the two smaller transcripts showed little or no change following heat shock. Run-on transcription assays in isolated maize nuclei showed a heat shock-induced increase in ubiquitin run-on transcripts that paralleled the increase in mature 2.1 kb mRNA levels over the first 3 h following the heat shock treatment. This result indicates that heat shock regulates ubiquitin gene expression at least in part at the transcriptional level. The subsequent rapid decline in steady-state mRNA levels, on the other hand, was not preceded by decreased ubiquitin gene transcription, raising the possibility of both transcriptional and posttranscriptional regulation. The run-on transcription assays also revealed a transient 5-fold reduction in rRNA gene transcription following heat shock, indicating that the transcriptional machinery for these genes is selectively sensitive to this stress.