Microarray analysis was initially performed to screen for differentially expressed genes between nitrate-and ammonium-fed rice (Oryza sativa L.) leaves. In total, 198 genes were shown to have a unique expression response to each treatment and most of the genes for which function is known were involved in signal transduction, plant stress resistance, transcriptional regulation, and basic metabolism. Northern blotting analysis confirmed that expression of the MT and PCS genes was highly upregulated in ammonium-fed leaves compared with expression in nitrate-fed leaves and it was further revealed that ammonium-fed leaves accumulated more cysteine and glutathione. The upregulated expressions of the MT and PCS genes and the higher levels of cysteine and glutathione in ammonium-fed leaves indicate that ammonium may be able to accelerate sulfur assimilation metabolism in rice leaves. Unexpectedly, Northern blotting analysis showed that the expression of the two key enzymes in the sulfur assimilation pathway, namely adenosine 5'-phosphosulfate reductase and O-acetylserine(thiol)lyase, was not upregulated by ammonium treatment. It was found that the total content of free amino acids was much higher in ammonium-fed leaves compared with nitrate-fed leaves, mainly resulting from an increase in several amino acids such as serine, asparagine, glutamine, and arginine. The increased amino acids, in particular serine (as a central substrate for the synthesis of the thiol metabolites), may have promoted sulfur assimilation metabolism under conditions of ammonium nutrition.