Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta, and it is highly conserved among eukaryotes. The rice PCNA promoter, truncated to position -263, was previously shown to confer meristematic tissue-specific expression in transgenic plants. By DNasel footprinting and gel retardation analysis, three cis-acting elements were defined in this truncated promoter, which designated site I (-201 to -194, CCAGGTGG), site IIa (-197 to -188, TGGGCCCGT) and site IIb (-178 to -169, TGGTCCCAC). Site I resembles the G-box, and sites IIa and IIb resemble the conserved motif (T/GGTCCCAT) that is found in promoter regions of auxin-regulated genes. Functional analysis of expression of a PCNA-GUS gene fusion in transgenic tobacco plants revealed that a mutation in site I in the full-length 2.0 kb promoter had no significant effect on the activity. However, the mutation in site I in the truncated -263 promoter, which had 14% of the activity of the full-length promoter, caused a considerable decrease in the activity, suggesting that site I contributes in part to transcriptional activation. Simultaneous disruption of sites IIa and IIb in the full-length promoter caused about 80-85% loss of promoter activity, while separate disruption of site IIa or site IIb resulted in no marked change on the activity. These observations suggest that site IIa and site IIb play an important role in the meristematic tissue-specific expression of the rice PCNA gene, presumably by mediating putative enhancer activities dependent on the far-upstream region.