This cross-sectional study of 226 HIV-positive Latino men and women sampled and assessed at an outpatient HIV clinic in Los Angeles examined the associations among acculturation, use of a substance before sex, and unsafe sexual behaviour. As acculturation increased, men and women were increasingly likely to have engaged in unsafe sex in the most recent sexual encounter since testing seropositive. In men, the association was partially mediated by use of a substance (primarily alcohol) in the three hours before the sexual encounter; in women, the association was not mediated by drug use. The findings underscore the need for culturally sensitive, secondary prevention programmes for HIV-positive persons.