Mature pollen from Petunia hybrida contains significant levels of 1-aminocyclopropane-1-carboxylic acid (ACC), and this ACC is thought to play a role in pollination-induced ethylene by the pistil. We investigated the developmental accumulation of ACC in anthers and pollen. The level of ACC in anthers was very low until the day before anthesis, at which time it increased 100-fold. A 1.1-kb partial ACC synthase cDNA clone (pPHACS2) was amplified from total RNA isolated from mature anthers by reverse transcriptase, followed by polymerase chain reaction using oligonucleotide primers synthesized to conserved amino acid sequences in ACC synthases. The expression of pPHACS2 mRNA during anther development was correlated with the accumulation of ACC and was localized to the pollen grain. The pPHACS2 cDNA was used to identify the PH-ACS2 gene from a library of genomic DNA fragments from Petunia hybrida. PH-ACS2 encoded an ACC synthase transcript of four exons interrupted by three introns. The ACC synthase protein encoded by the PH-ACS2 gene shared >80% homology with ACC synthases from tomato (LE-ACS3) and potato (ST-ACS1a). A chimeric PH-ACS2 promoter-β-glucuronidase (GUS) gene was used to transform petunia and transgenic plants were analyzed for GUS activity. GUS staining was localized to mature pollen grains and was not detected in other tissues. Despite similarities to LE-ACS3, we did not detect GUS activity under conditions of anaerobic stress or in response to auxin. A series of 5-prime-flanking DNA deletions revealed that sequences within the PH-ACS2 promoter were responsible for pollen-specific expression.