In this work we modified light illumination of the laser optoacoustic (OA) imaging system to improve the 3D visualization of human forearm vasculature. The computer modeling demonstrated that the new illumination design that features laser beams converging on the surface of the skin in the imaging plane of the probe provides superior OA images in comparison to the images generated by the illumination with parallel laser beams. We also developed the procedure for vein/artery differentiation based on OA imaging with 690 nm and 1080 nm laser wavelengths. The procedure includes statistical analysis of the intensities of OA images of the neighboring blood vessels. Analysis of the OA images generated by computer simulation of a human forearm illuminated at 690 nm and 1080 nm resulted in successful differentiation of veins and arteries. In vivo scanning of a human forearm provided high contrast 3D OA image of a forearm skin and a superficial blood vessel. The blood vessel image contrast was further enhanced after it was automatically traced using the developed software. The software also allowed evaluation of the effective blood vessel diameter at each step of the scan. We propose that the developed 3D OA imaging system can be used during preoperative mapping of forearm vessels that is essential for hemodialysis treatment.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.