The activity of the enzymes catalyzing the first two steps of sulfate assimilation, ATP sulfurylase and adenosine 5'-phosphosulfate reductase (APR), are confined to bundle sheath cells in several C(4) monocot species. With the aim to analyze the molecular basis of this distribution and to determine whether it was a prerequisite or a consequence of the C(4) photosynthetic mechanism, we compared the intercellular distribution of the activity and the mRNA of APR in C(3), C(3)-C(4), C(4)-like, and C(4) species of the dicot genus Flaveria. Measurements of APR activity, mRNA level, and protein accumulation in six Flaveria species revealed that APR activity, cysteine, and glutathione levels were significantly higher in C(4)-like and C(4) species than in C(3) and C(3)-C(4) species. ATP sulfurylase and APR mRNA were present at comparable levels in both mesophyll and bundle sheath cells of C(4) species Flaveria trinervia. Immunogold electron microscopy demonstrated the presence of APR protein in chloroplasts of both cell types. These findings, taken together with results from the literature, show that the localization of assimilatory sulfate reduction in the bundle sheath cells is not ubiquitous among C(4) plants and therefore is neither a prerequisite nor a consequence of C(4) photosynthesis.