The contrast agent Definity has recently been shown to have substantial nonlinear activity at high ultrasound frequencies (>10 MHz). In this study, measurements were performed to characterize the frequency dependent attenuation properties of Definity and populations of Definity that had been modified to preferentially isolate smaller bubbles through decantation or mechanical filtration. A narrowband pulse-echo substitution method was employed with a series of four transducers covering the frequency range from 2 to 50 MHz. "Native" Definity has peak in attenuation in the vicinity of 10 MHz and remains high until 50 MHz. This pattern is significantly different from other clinically approved agents and is consistent with recent reports of nonlinear activity at high frequencies. With increasing decantation times, the attenuation peak becomes more diffuse and occurs at progressively higher frequencies. By 3 h for example, attenuation continues to rise until 30 MHz. The bubble size distribution undergoes preferential skewing toward smaller bubbles with increasing decantation time. Between 30 s and 3 h, the mean bubble diameter goes from 3.99 to 0.98 micrometers. Mechanical filtration with 2 and 1 microm pores causes attenuation to rise until 15 and 40 MHz, respectively. Definity can therefore be manipulated to improve its relative activity at higher frequencies (>10 MHz), which has implications for ultrasound biomicroscopy and intravascular ultrasound applications. Further, these results suggest that agent handling can have a substantial impact on size distributions affecting lower frequency applications. Shell friction estimates derived from these data are lower than those reported for larger bubbles at lower frequencies.