Direct molecular evidence was obtained for the critical role of a single amino acid residue in a structural epitope distinguished by the monoclonal antibody MCA-13, which reacts selectively with severe isolates of citrus tristeza virus (CTV). Different CTV isolates cause a wide range of symptoms in the diverse citrus species they affect. Severe symptoms include decline, stem pitting, and seedling yellows. Plants infected by mild isolates are essentially symptomless. The monoclonal antibody MCA-13, which discriminates severe isolates from mild isolates of the virus, was used to map its epitope on the coat protein of CTV. A diverse group of coat protein genes of geographically and biologically distinct CTV isolates which are either MCA-13-reactive or MCA-13-nonreactive was cloned and sequenced. A series of mutant coat protein genes was constructed through oligonucleotide-directed, site-specific mutagenesis. The reactivity of the wild-type and mutant coat proteins expressed in Escherichia coli was evaluated by Western blotting using MCA-13 and polyclonal antibody prepared to CTV-coat protein. A single nucleotide alteration resulting in a Phe-->Tyr mutation at position 124 of the coat protein abolished the MCA-13 reactivity of a severe isolate, whereas a Tyr-->Phe mutation at the same site conferred MCA-13 reactivity on the coat protein of a previously nonreactive mild isolate of CTV.