Incorporation studies with C-13-, N-15-, and H-2-labeled substrates, followed by NMR analysis, show that the pyrimidine unit of thiamin (Vitamin B-1) originates from a C5N fragment, derived from C-2',2,N,C-6,5,5' of pyridoxol (Vitamin B-6) and an N-C-N fragment derived from L-histidine. Urocanic acid serves as an intermediate on the route of the N-C-N fragment of histidine into the thiamin pyrimidine.