PURPOSE: To assess the feasibility of percutaneous biopsy of low-conspicuity focal liver lesions with use of pulse-inversion harmonic imaging (PIHI) guidance in the late phase after injection of microbubble contrast agent. MATERIALS AND METHODS: Twelve patients referred for ultrasound (US)-guided biopsy had liver lesions lacking adequate conspicuity to undergo biopsy under conventional ultrasound (US) guidance. They underwent biopsy procedures performed with the use of PIHI guidance in the late phase after injection of Levovist. The conspicuity of lesions, procedure time, number of passes, and success and complication rates were documented and compared to a control group. The control group consisted of retrospective analysis of 19 patients who had undergone conventional US-guided biopsy procedures performed by the same radiologist. RESULTS: After contrast material injection, all lesions had sufficient increases in conspicuity to be targeted under PIHI guidance. Procedure time was prolonged in the PIHI group (66 minutes; range, 30-120 min; vs control, 33 min; range, 15-77 min; P <.01, Wilcoxon signed-rank test). The number of passes was greater in the PIHI group (1.9 +/- 0.3; vs control, 1.4 +/- 0.6; P <.01, chi(2) test). No statistically significant difference was observed when comparing success and complication rates. CONCLUSION: PIHI in the late phase after injection of Levovist generated clear delineation of focal liver lesions in all cases, allowing an accurate and safe sampling. This technique broadens the scope of US-guided biopsy to lesions lacking adequate conspicuity on conventional US.