considerable effort has been made towards understanding how acoustic and structural features contribute to emotional expression in music, but relatively little attention has been paid to the role of timbre in this process. Our aim was to investigate the role of timbre in the perception of affect dimensions in isolated musical sounds, by way of three behavioral experiments. In Experiment 1, participants evaluated perceived affects of 110 instrument sounds that were equal in duration, pitch, and dynamics using a three-dimensional affect model (valence, energy arousal, and tension arousal) and preference and emotional intensity. In Experiment 2, an emotional dissimilarity task was applied to a subset of the instrument sounds used in Experiment 1 to better reveal the underlying affect structure. In Experiment 3, the perceived affect dimensions as well as preference and intensity of a new set of 105 instrument sounds were rated by participants. These sounds were also uniform in pitch, duration, and playback dynamics but contained systematic manipulations in the dynamics of sound production, articulation, and ratio of high-frequency to low-frequency energy. The affect dimensions for all the experiments were then explained in terms of the three kinds of acoustic features extracted: spectral (e.g., ratio of high-frequency to low-frequency energy), temporal (e.g., attack slope), and spectro-temporal (e.g., spectral flux). High agreement among the participants' ratings across the experiments suggested that even isolated instrument sounds contain cues that indicate affective expression, and these are recognized as such by the listeners. A dominant portion (50-57%) of the two dimensions of affect (valence and energy arousal) could be predicted by linear combinations of few acoustic features such as ratio of high-frequency to low-frequency energy, attack slope, and spectral regularity. Links between these features and those observed in the vocal expression of affects and other sound phenomena are discussed.