The classic A1 locus in sweet pea (Lathyrus odoratus) was investigated by Bateson, Punnett, and Saunders in the early 20th century history of Mendelian genetics. The mutation, in the form of the pink and white cultivar ‘Painted Lady’, is known from the 18th century. We show that this locus is associated with a single base pair mutation (332 G/A) in the flavonoid 3′,5′-hydroxylase (F3′5′H) gene. This results in an amino acid change (111 glycine/aspartic acid) in the conserved substrate recognition site 1 of the enzyme. The mutant flower lacks the blue pigment delphinidin and is thus pink and white, rather than purple and blue as in the wild-type. This single amino acid change at a functionally important site appears to convert the enzyme from primary F3′5′H activity to a relatively efficient F3′H, as suggested by heterologous transformation into Arabidopsis PAP1D (a mutant line that produces anthocyanin constitutively).