Purpose: To identify a valid and reliable method to determine 40-km time trial (40K) performance in a laboratory setting. Methods: Part 1: Ventilatory threshold (VT) and 40K performance were determined on two occasions (February/September) using two subsets of cyclists (N = 15 each; (V) over dot O-2max 67.6 +/- 42/71.5 +/- 3.0 mL.kg(-1).min(-1)) to determine the predictive validity of VT assessments. Variables of interest were power output at VT, peak power output (MaxVT(w)), and average power output during 40K (40K(avgwatts)). For VT determination we used: breakpoint of VE/(V) over dot O-2; breakpoint of VENCO2; V-slope; RER = 1; and RER = 0.95. In part 2, test-retest reliability of VT and MaxVT(w) were examined in 20 subjects ((V) over dot O-2max 64.8 +/- 8.0 mL.kg(-1).min(-1)) on two occasions, separated by 48 h. Results: Regression analyses between power outputs at VTs and 40K(avgwatts) showed significant predictive validity for (February/September): V-slope (r = 0.79/0.84; SEE 15.5/13.3W), VE/(V) over dot O-2 (r = 0.80/0.81; SEE 15.2/14.2W), RER0.95. (r = 0.73/0.58; SEE 17.4/21.2W),RER1 (r = 0.75/0.74;SEE 16.8/16.7W),and MaxVT(w) (r= 0.81/0.73;SEE 15.0/17.1W). Paired t-tests between power outputs at VTs and the 40K(avgwatts) indicated that mean power outputs at VE/(V) over dot O-2 (261 29W; P = 0.33) and RER0.95 (274 +/- 55W; P = 0.93) in February and VE/(V) over dot O-2 (274 +/- 37W; P = 0.79) in September were not significantly different from the respective 40K(avgwatts) (277 +/- 30W/281 +/- 30W). Test-retest reliability analysis yielded the following intraclass correlation and relative test-retest errors: V-slope: 0.98, 2.6%; VE/(V) over dot O-2: 0.95, 5.3%; RER0.95: 0.87, 9.8%; RER1: 0.94,53%; VE/VCO2: 0.87, 12.1%; MaxVT(w): 0.98, 2.6%. Conclusion: The high test-retest reliability and consistent ability to accurately predict athletes' 40K(avgwatts) across a competitive season indicated that VE/(V) over dot O-2 was superior to the other evaluated methods.