The uptake of amino acids (measured with alpha-aminoisobutyric acid) by hepatocytes stimulated to proliferate by partial hepatectomy followed a biphasic pattern, with an initial peak 8-10 hr after surgery followed by a release of amino acids between 10 and 16 hr and a further increase between 16 and 20 hr after the operation. The second period of increased uptake coincided with the increase in DNA synthesis by the hepatocytes. Colchicine, and other microtubule disrupters administered at the time of partial hepatectomy, abolished the first period of increased amino acid uptake and prevented the cells from initiating DNA synthesis. Pulse-labeling experiments with alpha-aminoisobutyric acid revealed that colchicine did not inhibit amino acid transport per se, but prevented the increased capacity for amino acid transport induced by partial hepatectomy. An injection of colchicine 14 hr after hepatectomy prevented the release of amino acids by the liver and also substantially reduced the initiation of DNA synthesis. The data suggest that the microtubules may play a role in proliferative activation and also in the initiation of DNA synthesis of hepatocytes in vivo and that changes in amino acid transport are linked to these microtubule-requiring processes.