By means of two biological diffusion methods a penicillin content in various calf tissues was determined. For the injections PROKAIN Penicillin G Spofa, inj. adusum vet., was used in a single dose of 25 000 i. u. kg-1 body mass. For the tests three collection strains -- B. subtilis (ATCC 6633), Sarcina lutea (ATCC 9341), and Micrococcus flavus (ATCC 10240) were used. A quantitative determination of penicillin in the samples was accomplished by reading from the standard curves. Sensitivities of the two methods were approximately the same; Micrococcus flavus proved most suitable of the microorganisms employed. At 2, 3, 5 and 20 post-treatment days both test animals were positive; at 10 days three out of four calves examined were positive, whereas at 15 days two out of three calves examined were positive. The residues were most frequently found in the liver, kidney and at the injection sites. Maximum detectable concentrations were at the injections sites, minimum ones in the bone marrow. In the musculature (noninjection site) a small quantity of penicillin was found up to 20 days after the application. Current culinary and technological treatment of positive meat and liver samples did not always suffice for complete inactivation of the penicillin residues. For the tested preparation, a preslaughter withdrawal time of 30 days is recommended.