The predominant storage carbohydrates of mature carrot (Daucus carota L.) storage roots typically are the free sugars glucose and fructose. This trait is conditioned by the Rs allele. A naturally occurring recessive mutation, rs/rs, conditions a shift from these reducing sugars to sucrose. RT-PCR and sequencing revealed a unique 2.5 kb insert in the first and largest intron near the 5' end of the acid soluble invertase isozyme II gene of rs/rs carrots. This insert was not totally spliced out during mRNA processing. While the wild-type acid-soluble invertase isozyme II transcript (ca. 2 kb) was detected in Rs/Rs roots and leaves, none was observed in rs/rs roots throughout development. RT-PCR of rs/rs leaves revealed two novel transcripts (2.7 kb and 3.2 kb). A comparison of enzyme activity between the near-isogenic Rs/Rs and rs/rs carrot lines revealed very low acid-soluble invertase activity in rs/rs roots whereas neutral invertase, sucrose synthase and sucrose phosphate synthase levels were comparable. Those results and linkage analysis indicate that Rs is a candidate locus for carrot vacuolar acid-soluble invertase isozyme II. Although the 2.5 kb insert does not occur in the Rs wild-type acid-soluble invertase isozyme II allele, it does occur elsewhere in the genome of Rs/Rs plants.