Gallium-66 (T1/2 = 9.49 h) is an intermediate-lived radionuclide that has potential for positron emission tomography (PET) imaging of biological processes with intermediate to slow target tissue uptake. We have produced 66Ga by the 66Zn(p,n) 66Ga nuclear reaction using a small biomedical cyclotron and have investigated methods for purifying 66Ga that could be applied to the development of an automated processing system. Measured yields of 66Ga were very high with a production yield of nearly 14 mCi/μA-h at 14.5 MeV bombardment energy, a value in excellent agreement with theoretical predictions based on literature cross sections for the 66Zn(p,n) 66Ga reaction. Gallium-66 has been purified from irradiated zinc targets two ways, by cation-exchange chromatography and diisopropyl ether extraction. The concentrations of stable contaminants in 66Ga following the two processing methods were determined, and it was found that iron and zinc were present at levels up to an order of magnitude higher after cation-exchange chromatography. The bioconjugates DOTA-Tyr3-octreotide and DOTA-biotin were labeled with 66Ga purified by both methods. Following purification of 66Ga by solvent extraction, radiochemical yields in excess of 85% were obtained for both compounds, in contrast to much lower labeling yields (less than 20%) obtained after the cation-exchange separation. Higher concentrations of stable contaminants likely contributed to the poor radiochemical yields for labeling DOTA-Tyr3-octreotide and DOTA-biotin with cation-exchanged 66Ga. The lower purity and radiolabeling yields obtained using cation-exchange do not warrant the development of an automated processing system based on this method. Therefore, work is in progress to automate the diisopropyl ether extraction method for routine processing of 66Ga.