The ability to enhance specifically molecular markers of pathology with ultrasound has been previously demonstrated by our group employing a nanoparticle contrast agent. One of the advantages of this agent is it's relative nonechogenicity in the blood pool that allows increased contrast-to-noise between the blood pool and the bound, site-targeted agent. We sought to define the contrast agent concentration and acoustic parameters necessary to detect contrast enhancement in the blood so that molecular contrast enhancement could be more precisely defined. This study addresses two potential mechanisms that have been proposed for backscatter from the nanoparticle contrast agent in the blood pool - concentration-related scattering and phase conversion from liquid to gaseous perfluorocarbon. The nanoparticles were produced by methods currently standard in our laboratory using perfluorooctyl bromide (PFOB: b.p. 142° C) as the major component. Particle size was measured at 200±30 nm. Attenuation coefficient and backscatter of the agent were measured in whole porcine blood (hot 40%) and porcine plasma maintained at 37° C. Specimens were insonified using a broadband, single element transducer (5 MHz, 2.54 cm diameter, 5.08 cm focal length). Acoustic pulses with usable bandwidth of 1.5 to 10 MHz, a repetition rate of 1 kHz, and peak negative pressure of 3.9, 2.7, 1.5, and 0.8MPa (equivalent to M.I. of: 1.7, 1.2, 0.67, 0.36) were used to measure of attenuation coefficient and backscatter of nanoparticles at concentrations of 0.26, 0.51, 1.02, 2.04, 4.08×10 14 particles/mL while suspended in either whole porcine blood or porcine plasma. The attenuation coefficient was linear at all concentrations and power levels and shows no evidence of a resonant peak characteristic of liquid-to-gas phase conversion. The backscatter coefficient in plasma increased with concentration. However, in blood, backscatter was only significantly different from baseline at 2.04×10 14 particles/mL and above (8× the maximum anticipated dose). These data indicate that phase conversion of PFOB to gas is not the source of the contrast in molecular imaging with site targeted nanoparticles.