The effect of ethanol administration on the phosphorylation of thiamin, and on the transketolase activity (TKA) in erythrocytes, heart and liver, and on the thiamin excretion in urine was investigated in rats optimally supplied with thiamin over a period of 16 weeks. Ethanol ingestion resulted in a diminished concentration of thiamin (T), thiaminmonophosphate (TMP), thiamindiphosphate (TDP) and thiamintriphosphate (TTP) in blood and organs and in a reduced excretion rate of T in urine due to an impaired intestinal absorption. An ethanol induced alteration of the degree of thiamin phosphorylation became evident only after 16 weeks resulting in a decline of T and TMP in blood and of T in heart to undetectable amounts and in an enhancement of the TDP- and TTP-pool. Despite an increasing content of TDP in erythrocytes the TKA was lowered and the alpha-TK was enhanced during the test period suggesting a partially inhibited formation of the active holoenzyme by alcohol in vivo. However, the lowered TKA and the normal alpha-TK in liver are primarily suggestive of an apo-enzyme degeneration influenced by ethanol.