cDNA clones for rat liver 5-aminolevulinate synthase have been isolated and used to examine mRNA levels in different rat tissues. Northern hybridization analysis of total RNA from various rat tissues showed the presence of a single 5-aminolevulinate synthase mRNA species of estimated length 2.3 kilobases. Primer extension and RNase mapping studies indicated that the mRNA is identical in all tissues. Highest basal levels were seen in liver and heart. Administration of hemin to rats reduced the basal level of this mRNA only in liver but the heme precursor, 5-aminolevulinate (or its methyl ester), repressed the basal levels in liver, kidney, heart, testis, and brain. The drug 2-allyl-2-isopropylacetamide increased the mRNA level in liver and kidney only while human chorionic gonadotropin hormone elevated the level in testis. Administration of the heme precursor 5-aminolevulinate prevented these inductions. Nuclear transcriptional run-off experiments in liver cell nuclei showed that 2-allyl-2-isopropylacetamide and 5-aminolevulinate exert their effect by altering the rate of transcription of the 5-aminolevulinate synthase gene. The results indicate that a single 5-aminolevulinate synthase mRNA is expressed in all tissues and that its transcription is negatively regulated by heme.