Absorption, excretion and distribution of Benzyl Violet 4B were investigated in rats, deterimining the colour by the 2-wavelength technique. This colour was hardly absorbed when given orally; only 0.89% of the dose was recovered from the bile after 24 h. On the other hand, it was rapidly excreted through the bile when given intravenously; the cumulative recovery of biliary excretion amounted to 88.4% at 4 h and 95.9% at 24 h. The levels of the colour distributed in the liver, kidney abdominal muscle and blood serum were in the range of 1--3 microgram/g of tissue in rats fed a diet containing 5% Benzyl Violet 4B for 8 weeks, whereas they were slightly lower in rats fed the diet for 18 weeks. When rats were given the colour intravenously, there was no sex-related difference in the distribution of the colour in either Wistar or Sprague--Dawley rats, but the disappearance of the colour from the brain, liver, abdominal muscle, abdominal skin and ear of Sprague--Dawley rats was slower than from those of Wistar rats.