Lower mechanical index (MI) technique with newer microbubble agents has been introduced into clinical practice as a newer ultrasound (US) imaging. However, the efficacy in detecting tumour nodules has not been proven scientifically. The aim of this study was to elucidate the efficacy of a blood-pool image of real-time contrast-enhanced US under low MI in detecting liver tumours. 15 rabbits with VX-2 tumour were used; the number of implantations was none in two rabbits, one in four, two in five and three in four. US equipment was APLIO (Toshiba) with linear probe (3.5/7.0 MHz). The number, location and size of tumour nodules were examined by non-contrast tissue harmonic imaging (NC-US) or contrast-enhanced pulse subtraction harmonic imaging (C-US) under extra-low MI (MI 0.065) with the injection of Definity (30 microl kg(-1)). The number of tumour nodules detected by both NC-US and C-US were consistent with the histopathological results in five rabbits - two with none, two with one nodule and one with two nodules. In the other 10 rabbits, C-US showed all the implanted tumours and small daughter nodules around them that were confirmed by histopathology. However, NC-US failed to demonstrate two implanted nodules and all the daughter nodules. On the basis of the histopathological results, detectability of implanted tumour was not significantly different between NC-US (24/26, 92.3%) and C-US (26/26, 100%). However C-US was superior to NC-US in delineating the nodules and in detecting small daughter nodules. The sizes of the implanted tumour nodules measured by histopathology correlated closely with those measured by C-US. Real-time blood-pool images by pulse subtraction harmonic imaging under extra-low MI with Definity will contribute to the improvement of the ultrasound delineation and detection of liver tumours.