ABSTRACT In vitro gamma interferon release assays (IGRAs) are increasingly used as an alternative to the traditional tuberculin skin test for the diagnosis of latent Mycobacterium tuberculosis infection. Evaluation of the QuantiFERON-TB Gold in-tube assay (QFT-IT) prior to large-scale implementation at the Stanford Hospital and Clinics for a health care worker screening program revealed a critical preanalytical factor affecting the results. We found that incubation delay significantly increased the frequency of indeterminate results. In this study, QFT-IT was performed with samples from healthy volunteers, and replicate tubes were incubated at 37°C either immediately or after a delay at room temperature for 6 and 12 h. No indeterminate results (0/41) were seen when the assay was performed with immediate incubation. Incubation delays of 6 and 12 h yielded indeterminate results at rates of 10% (2/20) ( P = 0.10) and 17.1% (7/41) ( P = 0.01), respectively. The increased rate of indeterminate results was due to a decrease in the mean values for the mitogen-nil tubes when incubation was delayed for 6 h ( P = 0.004) and 12 h ( P < 0.001). The rates of concordance of positive or negative results obtained following immediate incubation and following 6- and 12-h delays were 77.8% (14/18) and 79.4% (27/34), respectively. Subsequent implementation of the immediate incubation procedure in our screening program for 14,830 health care workers yielded an indeterminate result rate of 0.36% over a period of 12 months, a significant improvement over the reported rates of 5 to 40% for QFT-IT. We conclude that immediate incubation of QFT-IT tubes is an effective way to minimize indeterminate results. The effect of incubation delay on the accuracy of QFT-IT remains to be determined.