The effects of soil or <em>in vitro</em> grown plants, pretreatment conditions, donor tissue and isolation procedure on protoplast yield from cotyledons and leaves of tomato cv. 'Perkoz' and 'Zorza' were studied. The highest protoplast yield of 1.5 x 10<sup>7</sup>/g FW was obtained from leaves of <em>in vitro</em> grown plants. Low light intensity during donor plants <em>in vitro</em> culture and dark pretreatment were essential for successful protoplast isolation while cold pretreatment was not. Tissue preplasmolysis prior to transfer to enzyme mixture increased 4-fold the number of isolated protoplasts. Glycine and bovine serum albumin in the isolation medium did not significantly influence the protoplast yield.