The dependency of the timbre of musical sounds on their fundamental frequency (F0) was examined in three experiments. In experiment I subjects compared the timbres of stimuli produced by a set of 12 musical instruments with equal F0, duration, and loudness. There were three sessions, each at a different F0. In experiment II the same stimuli were rearranged in pairs, each with the same difference in F0, and subjects had to ignore the constant difference in pitch. In experiment III, instruments were paired both with and without an F0 difference within the same session, and subjects had to ignore the variable differences in pitch. Experiment I yielded dissimilarity matrices that were similar at different F0’s, suggesting that instruments kept their relative positions within timbre space. Experiment II found that subjects were able to ignore the salient pitch difference while rating timbre dissimilarity. Dissimilarity matrices were symmetrical, suggesting further that the absolute displacement of the set of instruments within timbre space was small. Experiment III extended this result to the case where the pitch difference varied from trial to trial. Multidimensional scaling (MDS) of dissimilarity scores produced solutions (timbre spaces) that varied little across conditions and experiments. MDS solutions were used to test the validity of signal-based predictors of timbre, and in particular their stability as a function of F0. Taken together, the results suggest that timbre differences are perceived independently from differences of pitch, at least for F0 differences smaller than an octave. Timbre differences can be measured between stimuli with different F0’s.