PURPOSE: To evaluate the psychometric characteristics of the Research on Urban Trail Environments (ROUTES) Trail Use Questionnaire. DESIGN: Test-retest reliability was assessed by repeated measures (study 1); validity was assessed by comparing reported trail use to self-reported and objectively measured physical activity (PA) levels (study 2). SETTING: Study 1: a religious institution situated near a Los Angeles trail. Study 2: 1-mile buffer zones surrounding three urban trails (Chicago, Dallas, and Los Angeles). SUBJECTS: Thirty-four adults between 40 and 60 years of age (10 men and 24 women) completed the ROUTES questionnaire twice (study 1). Study 2 participants were 490 adults (48% female and 73% white), mean age 48 years. MEASURES: Trail use for recreation and transportation purposes, time and distance spent on trails, and characteristics of the trail and other trail users. PA was measured using the International Physical Activity Questionnaire and accelerometry. ANALYSES: Pearson correlation coefficients and kappa statistics were used for test-retest reliability for continuous and categorical variables, respectively. Generalized linear models were used to evaluate hypotheses on PA comparing trail users and nonusers. RESULTS: Test-retest statistics were acceptable (kappa = .57, r = .66). Validity was supported by correlations between indices of trail use with self-reported PA and accelerometry, and significant group differences between trail users and nonusers in PA levels. CONCLUSIONS: The ROUTES Trail Use Questionnaire demonstrated good reliability and validity.