The contrast agent Definity has recently been shown to have substantial nonlinear activity at high ultrasound frequencies (>10 MHz), which has been hypothesized to be associated with the presence of a significant subpopulation of smaller bubbles (<1-2 micrometers). In this study, we characterized the frequency dependant attenuation properties of Definity and populations of Definity that had been modified to preferentially isolate smaller bubbles through decantation or mechanical filtration procedures. A narrowband pulse-echo substitution method was employed with a series of three transducers covering the frequency range from 2 to 50 MHz. 'Native' Definity was found to have a peak in attenuation in the vicinity of 10 MHz and remained 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 became more diffuse and occurred at progressively higher frequencies. By 3 hours for example, attenuation continued to rise until 30 MHz. The bubble size distribution, as measured with a coulter counter, underwent preferential skewing towards smaller bubbles with increasing decantation time. Between 30 seconds and 3 hours, the mean bubble diameter went from 3.99 to 0.98 micrometers. Mechanical filtration with 2 and 1 micrometer pores resulted in attenuation that rose until 15 and 40 MHz respectively. This demonstrates that Definity populations can be manipulated to have improve activity at higher frequencies (>10 MHz), which has implications for UMB and IVUS applications.