Nitrogen nutrients can regulate anthocyanin biosynthesis in Arabidopsis thaliana. In this investigation, we report the nitrogen regulation of anthocyanin biosynthesis activated by TTG1-GL3/TT8-PAP1 in red pap1-D cells. To understand the mechanisms of nitrogen regulation, we employed red pap1-D cells and wild-type cells (as a control) to examine the effects of different nitrogen treatments on anthocyanin biosynthesis. In general, the higher concentrations of ammonium and high total nitrogen tested (e.g., 58.8 and 29.8 mM total nitrogen consisting of NH(4)NO(3) and KNO(3)) reduced the levels and molecular diversity of anthocyanins; in contrast, the lower concentrations of ammonium and total nitrogen conditions (e.g., 9.4 mM KNO(3) and the depletion of nitrogen) increased the levels and molecular diversity of anthocyanins. An expression analysis of the main regulatory and pathway genes showed that at conditions of higher concentrations of ammonium and total nitrogen, the expression levels of PAP1 and TT8 decreased, but the expression levels of LBD37, 38 and 39, three negative regulators of anthocyanin biosynthesis, increased. In addition, the expression levels of the main pathway genes decreased. In contrast, at conditions of lower concentrations of ammonium and total nitrogen, the expression levels of PAP1, TT8 and the main pathway genes increased, whereas those of LBD37, 38 and 39 decreased. These results show that nitrogen regulation of anthocyanin biosynthesis in red cells undergoes a mechanism by which nitrogen controls the expression of genes encoding both main components of the TTG1-GL3/TT8-PAP1 complex and negative regulators. Based on these observations, we propose that the regulatory mechanism of nitrogen may occur via two pathways to control the expression of genes encoding positive and negative regulators in red pap1-D cells.