Sonazoid is a new contrast agent for ultrasound imaging comprising an aqueous suspension of lipid-stabilised perfluorobutane (PFB) gas microbubbles. A respiration-metabolism chamber system was developed to collect exhaled air following intravenous administration of Sonazoid to rats. Analysis of PFB in the exhaled rat air was performed using a modified version of an earlier published method for blood samples, i.e. an automatic headspace gas chromatographic mass spectrometric (GC-MS) method using electron impact ionisation. The calibration standards were PFB diluted in air (2.5-1800 pg/ml). Perfluoropentane (PFP) was used as an internal standard and the MS detector was set to single ion monitoring of the base fragment ions of PFB (m/z 69 and 119) and PFP (m/z 69). The calibration curve, made by plotting the peak area ratios of PFB (m/z 69) to PFP (m/z 69) against the theoretical concentration of PFB, was fitted to a linear equation with weighting 1/y2 and found to be reproducible. The lower limit of quantification (LLOQ) was 2.5 pg PFB/ml. The between-day variation of the method was below 2.6% relative standard deviation (R.S.D.) and the within-day variation of the method was below 6.4% R.S.D. The accuracy of the method was evaluated and showed a relative error less than 5.2%. PFB was found to be stable for 14 days when stored in Tedlar sample bags at room temperature. An even lower detection limit may be obtained by using the more time-consuming process of solid-phase micro extraction; thus, by concentrating PFB on carboxen-PDMS fibres an LLOQ of 0.5 pg PFB/ml was obtained. When five rats were given an i.v. bolus injection of Sonazoid at a dose of 8 microl microbubbles/kg a mean recovery of 96% (range, 81-110%) was found during 24 h; more than 50% was exhaled during the first 30 min after injection.