In recent years, our knowledge of the behaviour of ultrasonic microbubble contrast agents has improved substantially through in vitro experiments. However, there has been a tendency to use high concentrations of contrast agents in suspension, so that ultrasonic backscatter data are generated by a cloud of microbubbles. Such experiments involve a variety of assumptions with validity that is open to question. In addition, high concentrations of microbubbles cannot be used to understand the behaviour of individual microbubble scatterers. This paper proposes a technique that minimises the number of assumptions that need to be made to interpret in vitro experimental data. The basis of the technique is a dilute suspension of microbubbles that makes single scattering events distinguishable. A commercial scanner was used to collect radio frequency (RF) data from suspensions of two different contrast agents, Quantison(TM) and Definity. Backscatter data were collected over a range of acoustic pressures. It was found that Definity provided a constant number of scattering events per unit volume of suspension for almost all applied acoustic pressures. Quantison(TM) demonstrated an increasing number of scattering events per unit volume with increasing acoustic pressure. Below 0.6 MPa, QuantisonTM scatterers were not individually detectable and provided levels of backscatter similar to those of a blood-mimicking fluid, which suggests that QuantisonTM microbubbles had almost linear scattering behaviour. At acoustic pressures greater than 0.6 MPa, both agents appeared to provide echoes from free bubbles. The change in the number of scatterers per unit volume with acoustic pressure cannot be demonstrated using high concentrations of contrast agent. (C) 2003 World Federation for Ultrasound in Medicine Biology.