A differential mechanical technique for tissue separation, based on the different physical resistance to grinding between mesophyll (M) and bundle sheath (BS) cells, was tested on dicotyledonous C4 plants A triplex canscens, A triplex halimus, Gomphrena globosa, Amaranthus retroflexus, Amaranthus caudatus and Portulaca oleracea. A metal sieve (35 mesh) was placed inside a mortar and pieces of leaves (0.5 cm2) were ground in an aqueous medium on the sieve to obtain a homogenate. The homogenate was at first collected below the sieve and was then filtered through six layers of muslin. Microscopic examination showed that the filtrate was enriched by the M cells and the residue was enriched by BS cells, few of which were broken. The BS cell fraction was then vigorously ground and filtered; this second filtrate was named the BS cell fraction and the first filtrate was named the M cell fraction. Ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) (RuBPC) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEPC) were assayed, and chlorophyll determinations and protein estimations were made on both fractions. As expected, PEPC showed higher activities in the M fractions; contrary to expectation RuBPC was present in M cell fractions in the six dicotyledonous C4 plants tested. The relative high RuBPC activities found in the M fraction could not be explained in terms of bundle sheath contamination.