Expression of MdACS1, coding for 1-aminocyclopropane-1-carboxylate synthase (ACS), parallels the level of ethylene production in ripening apple (Malus domestica) fruit. Here we show that expression of another ripening-specific ACS gene (MdACS3) precedes the initiation of MdACS1 expression by approximately 3 weeks; MdACS3 expression then gradually decreases as MdACS1 expression increases. Because MdACS3 expression continues in ripening fruit treated with 1-methylcyclopropene, its transcription appears to be regulated by a negative feedback mechanism. Three genes in the MdACS3 family (a, b, and c) were isolated from a genomic library, but two of them (MdACS3b and MdACS3c) possess a 333-bp transposon-like insertion in their 5# flanking region that may prevent transcription of these genes during ripening. A single nucleotide polymorphism in the coding region of MdACS3a results in an amino acid substitution (glycine-289 / valine) in the active site that inactivates the enzyme. Furthermore, another null allele of MdACS3a, Mdacs3a, showing no ability to be transcribed, was found by DNA sequencing. Apple cultivars homozygous or heterozygous for both null allelotypes showed no or very low expression of ripening-related genes and maintained fruit firmness. These results suggest that MdACS3a plays a crucial role in regulation of fruit ripening in apple, and is a possible determinant of ethylene production and shelf life in apple fruit.