This study describes the process of developing and validating the Sexual Prejudice in Sport Scale (SPSS), which is a multidimensional instrument developed to assess attitudes toward lesbians and gay men (LG) in sports. The authors conducted two studies: first, to establish the factor structure of the SPSS on 297 heterosexual athletes; and, second, to test the reliability and validity of the resulting 19-item scale on a sample of 311 heterosexual and 160 LG athletes. Exploratory factor analysis of an initial item pool yielded three factors: open rejection, which assesses the blatant prejudice expressed toward LG people; denial of visibility, which evaluates attitude toward the coming out of LG people; and gendering performance, which corresponds to gender stereotypes about performance/skills of LG people. A confirmatory factor analysis was conducted to assess the stability of the SPSS. The authors documented internal consistency, test-retest stability, and convergent/divergent validity. Implications and directions for future research are discussed.