The effect of Chloroethylthiamin (CET), a structural analog of thiamin, on thiamin pyrophosphokinase (TPKase) activity of the supernatant of rat isolated enterocytes was compared with that on thiamin intestinal transport by rat everted jejunal sacs. Thiamin- thiazol-2(14C) was used as a substrate both for TPKase activity and for thiamin serosal transport and uptake. CET strongly inhibited TPKase activity of isolated enterocytes: at molar concentrations 10 or 100 times higher than labeled thiamin, the inhibition was 57 and 100% respectively. The inhibition was of the competitive type, with a Ki = 15 microM. At a molar concentration 10 times higher than labeled thiamin, CET lowered the thiamin serosal transport by 60%, and the sac wall content of free and phosphorylated thiamin by 54 and 42% respectively. At the 1:10 thiamin: CET molar ratio, the extent of the reductions of TPKase activity (57%) and of phosphorylated thiamin content of intestinal sac walls (42%) were of the same order. This indicates a relationship between the two events. Moreover, since TPKase activity inhibition alone resulted in the lowering of labeled thiamin serosal transport, thiamin phosphorylation and transport are probably two strictly related processes.