Abstract: ITG ⁶⁸Ge/⁶⁸Ga generator allows for dilute HCl to effectively elute ⁶⁸Ga³⁺ without metal impurities. The impact of the optimal elution time, the best peak segment and the effects of different buffers on the imaging and biodistribution of the ⁶⁸Ga were explored with the ITG ⁶⁸Ge/⁶⁸Ga generator. According to the manufacturer protocol, a 740 MBq self-shielded ⁶⁸Ge/⁶⁸Ga generator was eluted with 4 mL of 0.05 mol/L HCl. After the beginning of elution, the generator was eluted again at 2, 4, 6, 8, 10, 12, 14 hours to record radioactivity. The ⁶⁸Ga was eluted with HCl in each fraction of 0.5 mL, then eluted with every 0.1 mL in the dense fractions and the radioactivity was recorded. MicroPET imaging and biodistribution were performed in normal mice after adjusting the pH of ⁶⁸Ga³⁺ solution with sodium acetate, NaOH and HEPES separately. The results showed that 4 hours post elution, 91% of the maximum achievable radioactivity was accumulated. The best peak was at 2.3 mL and the radioactivity of the top fraction (0.1 mL) was 22 MBq. The uptake of ⁶⁸Ga³⁺ radioactivity was 6.9%ID/g in the blood at 1 hour post injection, suggesting that purity of a tracer was essential because free ⁶⁸Ga³⁺ may affect the contrast of the image and dection of tumors. The radioactivity uptake of the bladder was high, suggesting that the ⁶⁸Ga³⁺ was metabolized by the urinary system. There was no significant difference in the ⁶⁸Ga³⁺ imaging and biodistribution as using sodium acetate, HEPES and NaOH as buffer solution (P>0.05). The results showed that ITG ⁶⁸Ge/⁶⁸Ga generator was simple and convenient for manual operation. The generator could be eluted every 4 hours and the best peak segment was at 2.3 mL. Sodium acetate seems to be the most appropriate buffer in clinic.