Conventional gray-scale myocardial contrast echocardiography cannot distinguish perfused but attenuated from nonperfused myocardium because both may appear similar at low image intensity. We hypothesized that with radiofrequency spectral analysis of attenuated ultrasound signals, the harmonic-to-fundamental frequency ratio of the peak power spectrum (HFRp) could determine the presence of contrast microbubbles. We measured frequency responses of Optison microbubbles at defined degrees of ultrasound signal attenuation with different formulations of silicone (55D, 80A, and 3M); gray-scale intensities of Optison plus water compared with degassed water were analyzed at different attenuation settings (-25, -32, and -44 dB, respectively). HFRp values of Optison plus water were significantly higher than reference values of degassed water at each attenuation setting (55D, -14 +/- 2 dB versus -30 +/- 2 dB, P < .001; 80A, -19 +/- 2 dB versus -30 +/- 3 dB, P < .01; 3M, -22 +/- 2 dB versus -30 3 dB, P < .05), even though conventional videodensitometric analysis could not distinguish them. HFRp analysis objectively detects microbubbles in clinically relevant conditions of attenuation.