Background: Comprehensive assessment of sedentary behavior (SB) and physical activity (PA), including transport-related activities (TRA), is required to design innovative PA promotion strategies. There are few validated instruments that simultaneously assess the different components of human movement according to their context of practice (e.g. work, transport, leisure). We examined test-retest reliability and validity of the Sedentary, Transportation and Activity Questionnaire (STAQ), a newly developed questionnaire dedicated to assessing context-specific SB, TRA and PA. Methods: Ninety six subjects (51 women) kept a contextualized activity-logbook and wore a hip accelerometer (Actigraph GT3X + TM) for a 7-day or 14-day period, at the end of which they completed the STAQ. Activity-energy expenditure was measured in a subgroup of 45 subjects using the double labeled water (DLW) method. Test-retest reliability was assessed using intra-class-coefficients (ICC) in a subgroup of 32 subjects who filled the questionnaire twice one month apart. Accelerometry was annotated using the logbook to obtain total and context-specific objective estimates of SB. Spearman correlations, Bland-Altman plots and ICC were used to analyze validity with logbook, accelerometry and DLW data validity criteria. Results: Test-retest reliability was fair for total sitting time (ICC = 0.52), good to excellent for work sitting time (ICC = 0.71), transport-related walking (ICC = 0.61) and car use (ICC = 0.67), and leisure screen-related SB (ICC = 0.64-0.79), but poor for total sitting time during leisure and transport-related contexts. For validity, compared to accelerometry, significant correlations were found for STAQ estimates of total (r = 0.54) and context-specific sitting times with stronger correlations for work sitting time (r = 0.88), and screen times (TV/DVD viewing: r = 0.46; other screens: r = 0.42) than for transport (r = 0.35) or leisure-related sitting-times (r = 0.19). Compared to contextualized logbook, STAQ estimates of TRA was higher for car (r = 0.65) than for active transport (r = 0.41). The questionnaire generally overestimated work-and leisure-related SB and sitting times, while it underestimated total and transport-related sitting times. Conclusions : The STAQ showed acceptable reliability and a good ranking validity for assessment of context-specific SB and TRA. This instrument appears as a useful tool to study SB, TRA and PA in context in adults.