The 5'-terminal 31 nucleotides of the 146-nucleotides-long 5' noncoding region of plum pox potyvirus (PPV) are highly conserved in all the members of the Potyvirus genus. To map the sequences of the 5' noncoding region that are necessary in vivo for infectivity, we have constructed a nested set of substitution and deletion mutants. While we were not able to infect Nicotiana clevelandii plants with full-length PPV transcripts bearing mutations in the 5'-terminal 35 nucleotides of the viral genome, the deletion of long sequences located between nucleotides 39 and 145 did not alter either the rate of infection or viral accumulation. Nevertheless, these mutants were not able to compete with the wild-type strain in coinoculation experiments. Plants infected with a PPV mutant that lacked nucleotides 127 to 145 showed a very mild symptomathology; the wild-type symptom severity was recovered after spontaneous second-site mutations.