Homophobia and heterosexism in women's athletics have been studied extensively using a qualitative approach. Limited research from a quantitative approach has been conducted in the area and none with a sport-specific instrument. The purpose of the current study was to develop a valid and reliable questionnaire to measure heterosexist attitudes in women's collegiate athletics based on the tripartite model of attitudes (Zanna & Rempel, 1988). A panel of experts analyzed a pool of 40 items for item-content related validity. Two samples of collegiate female athletes completed the second and third versions of the Heterosexist Attitudes in Sport-Lesbian questionnaire. Initial evidence of construct validity was found for a 14-item measure of heterosexist attitudes with four subscales: (1) Cognitive/Affective, (2) Language Behaviors, (3) Inclusion Behaviors, and (4) Avoidance of the Lesbian Label. Composite reliability of the four subscales ranged from 0.64 to 0.84. (Contains 4 tables and 2 figures.)