We report the validation of a reversed-phase gradient HPLC method allowing simultaneous quantification of retinol, retinyl esters, tocopherols and selected carotenoids in lung, liver and plasma of mouse, rat and guinea pig (gp) using a diode array detector. A significant species difference was observed regarding the distribution of retinol and retinyl esters. The levels of total retinol in lung, liver and plasma were in the following order: mouse ≫ rat > gp; rat >mouse > gp; and gp ≫ rat > mouse, respectively. Furthermore, comparison studies revealed similarities between the vitamin A profiles of human and gp lung samples.