Guppies are a model vertebrate for studies of sexual selection and life history evolution. None the less, there have been few investigations of the factors responsible for maintaining extreme within-population genetic variation in male coloration. In a laboratory study, we tested the hypothesis that frequency-dependent mate choice contributes to the maintenance of this variation. We attempted to avoid biases inherent in earlier studies of the 'rare male effect' by familiarizing females to males bearing a particular colour pattern and later presenting them with alternate male types, in equal numbers. Females were significantly more likely to mate with males having novel colour patterns than with males having a colour pattern with which they were familiar. This result is consistent with the hypothesis that mate choice is frequency dependent. Other factors such as male and female size were unrelated to mate preference. Implications of the results for theories of sexual selection and the maintenance of variation are discussed. Copyright 1999 The Association for the Study of Animal Behaviour.