A study was made of solo and ensemble performance of the same musical material as related to systems of intonation postulated by certain acoustical, musical, and psychological theories. In particular, it was desired to check earlier findings that unaccompanied performance and listener preferences approximate Pythagorean intonation and to extend a similar line of investigation to ensemble performance.Solo and ensemble performances by 24 well‐trained string quartet players were recorded from which stratified random samples of tones were obtained for frequency analysis. This analysis was made through the use of 16‐mm sound‐on‐film loops with a chromatic stroboscope (Stroboconn).The results confirm earlier findings for unaccompanied melodies and indicate that Pythagorean intonation is also most typical of ensemble performance. This tendency appears to dominate any “cultural conditioning” which may exist for equi‐tempered intonation.