This study examined the extent of measurement invariance of the Basic Psychological Needs in Exercise Scale responses (BPNES; Vlachopoulos & Michailidou, 2006) across male (n = 716) and female (n = 1,147) exercise participants. BPNES responses from exercise participants attending private fitness centers (n = 1,012) and community exercise programs (n = 851) were used. The 3-factor BPNES confirmatory factor analysis model, discriminant validity, and scale reliability were supported for both male and female participants separately. The multisample models supported the configural invariance, partial metric invariance, partial measurement error invariance, and partial scalar invariance of the BPNES responses across gender. Both male and female participants attached the same meaning to the constructs assessed by the BPNES items. The BPNES score invariance properties support tests of the needs universality hypothesis offered by self-determination theory across gender in exercise and meaningful comparison of the autonomy, competence, and relatedness construct latent means across gender. (Contains 4 tables.)