Previous studies indicated that a DNA fragment comprising nucleotides (nt) -164 to +45 of the RTBV promoter is sufficient to drive phloem-specific expression of a reporter gene in transgenic rice plants. In addition, two potential cis elements, Box I (nt -3 to +5) and Box II (nt -53 to -39) were identified by DNA-protein interaction assays. In this study, the results of further in vivo studies involving mutagenesis of selected DNA sequences and analysis of expression of a reporter gene in transgenic rice plants revealed that, in addition to Box I and Box II, other elements are required for phloem-specific gene expression, among which are a direct repeat (ASL Box, nt -98 to -79) and a GATA motif (nt -143 to -135). All the these elements bind rice nuclear factors specifically, and mutations of the elements were identified that resulted in loss-of-competition in electrophoretic mobility shift assays. A DNA fragment comprising nt -164 to -32, which contains Box II, the ASL Box and the GATA motif, conferred phloem-specific reporter gene expression independent of Box I when fused to a heterologous CaMV 35S minimal promoter and introduced to transgenic rice plants. Studies that introduced point mutations suggested that in the context of the -103 to +45 promoter fragment, Box II and the ASL Box act synergistically to confer tissue-specific gene expression. Similar studies in the -164 to +45 promoter fragment indicated that the -164 to -103 region, which includes the GATA motif, contains sequences that are functionally redundant with those in the -103 to -32 region, including the ASL Box and Box II. It is concluded that these regions act additively to direct phloem-specific gene expression.