Plant stigmasterol possesses many therapeutic properties, and it is used as one of the precursors for synthesis of synthetic progesterone. In this study, the stigmasterol content was analyzed in various plant parts of tomato (Solanum lycopersicum), and increased synthesis was attempted in tomato tissue culture cells. Stigmasterol content varied among the plant parts tested and the varieties studied. Leaves were found to have high stigmasterol in all of the varieties. Expression analysis of stigmasterol biosynthetic pathway genes revealed that the levels of smt2, dwf5, and dwf1 expression were highly variable among different organs and varieties. Expression of ste1and smo2 was below the detectable level in most tissues. Except in the leaves of variety S-22 and the stem and roots of variety PKM-1, expression of CYP710A11 was relatively uniform. Overexpression of CYP710A11 in tomato callus by the Agrobacterium tumefaciens transformation method resulted in a 2.6-fold increase in stigmasterol. © 2016, The Society for In Vitro Biology.