We compared perception of mistuned intervals in unaccompanied melodies performed by trumpet, violin, and voice, and examined whether there were differences between the three timbres in performances with and without vibrato. Participants were 144 university music students. Listeners heard the three unaccompanied solo performers in two vibrato conditions (with and without vibrato), and three intonation conditions (selected melodic intervals were in tune, sharp 25 cents, or flat 25 cents relative to equal temperament). All three stimuli were perceived as more out of tune when there was no vibrato compared to vibrato. In performances without vibrato, violin was judged as more out of tune than voice and trumpet across all three tuning conditions. Melodies performed with vibrato were judged differently: Violin was judged as least in tune for intervals mistuned in the flat direction, trumpet was heard as least in tune for intervals mistuned sharp, and voice was judged least in tune when intervals were in tune (relative to equal temperament). Differences in perception between timbres may be influenced by characteristics of the vibrato itself such as modulation width, rate, and type.