A simple, efficient, and economical method is presented for the preparation of radioactive betaine. It involves the incubation of radioactive choline with osmolyte-free Pseudomonas aeruginosa previously grown in hyperosmolar medium with choline as an osmoprotectant. The summarized procedure was as follows: (i) bacteria were grown in high P-i basal salt medium (HPi-BSM) with 20 mM succinate, 18.7 mM NH4Cl, 0.8 M NaCl, and 1 mM nonradioactive choline. After the bacterial pellet was obtained, it was suspended in deionized water to release osmolytes accumulated during growth; (ii) suspension of the pellet, free of osmolytes, in hyperosmolar HPi-BSM with [methyl-C-14]-choline (55 nCi/nmol) without the carbon and nitrogen sources. Incubation of the mixture at 37 degrees C for 8-30 h. When only 10% of the initial radioactivity remained in the supernatant, it was withdrawn after centrifugation and the pellet suspended in deionized water. This step released the accumulated betaine plus some contaminants. Purification of betaine contained in the aqueous supernatant was carried out after rotoevaporation to dryness and solubilization of the residue in methanol. The methanolic extract was rotoevaporated to dryness, the residue solubilized in 10% acetic acid and transferred to a Dowex 50-X8 column. After the column was washed with water and 2 M NH4OH, betaine was eluted by the addition of 4 M NH4OH. The total procedure for obtaining pure radioactive betaine resulted in a yield of 80%. The product obtained was chemically and radiochemically pure, with a specific radioactivity of 54 i: I nCi/nmol.