Sound calibration is employed in many commercial audio systems for improving sound quality. This process includes the estimation of the room transfer function (RTF) between each loudspeaker and a microphone located at the listeners' position. Current methods for RTF estimation employ calibration signals, such as noise or tones, in a dedicated process applied to each loudspeaker separately. Such an estimation disrupts normal playback, is time consuming, and requires user intervention. A perceptuallytransparent online RTF estimation method for a two-channel system, which employs calibration signals generated using the original audio signals and complementary filters, is proposed in this article. These calibration signals are uncorrelated across the two channels, which facilitates the online estimation of both channels using a single microphone. Estimation performance is investigated for an experimental system and displays low estimation errors. Finally, a subjective evaluation via a listening test shows that playback of calibration signals is perceptually-transparent under some of the conditions investigated.