The preparation and stability of lysozyme labeled with ³H or ¹³¹I were studied. ¹³¹I-lysozyme was found to be convenient for investigation on biotransformation of the enzyme. The transport of ¹³¹I-lysozyme across rat intestine in vitro was studied using rat everted jejunum. The transport of lysozyme from the mucosal to serosal side was confirmed by several methods such as TCA-precipitation, immunoprecipitation and enzymatic activity assay. The intestinal absorption of ¹³¹I-lysozyme in vivo was studied in rats paying attention to the lymphatics and the portal vein as the route of absorption. The absorption rate of immunoprecipitable ¹³¹I via lymphatics was 0.07% of the dose during the first 6 hr after intraintestinal administration of 2 mg/kg of ¹³¹I-lysozyme. The absorption rate of immunoprecipitable ¹³¹I via portal vein was calculated as 1.99% of ¹³¹I-lysozyme dosed, by pharmacokinetic analyses on the concentration of immunoprecipitable ¹³¹I in serum. Absorption via lymphatics was 3.24% of the total immunoprecipitable ¹³¹I absorbed via both routes. The main route in intestinal absorption of ¹³¹I-lysozyme following intraintestinal administration was suggested to be the portal vein, not lymphatics.