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We developed novel plasmids and T-DNA binary vectors that incorporate a modified and more useful form of the superpromoter. The superpromoter consists of a trimer of the octopine synthase transcriptional activating element affixed to the mannopine synthase2# (mas2#) transcriptional activating element plus minimal promoter. We tested a superpromoter-bglucuronidaseA fusion gene in stably transformed tobacco (Nicotiana tabacum) and maize (Zea mays) plants and in transiently transformed maize Black Mexican Sweet protoplasts. In both tobacco and maize, superpromoter activity was much greater in roots than in leaves. In tobacco, superpromoter activity was greater in mature leaves than in young leaves, whereas in maize activity differed little among the tested aerial portions of the plant. When compared with other commonly used promoters (cauliflower mosaic virus 35S, mas2#, and maize ubiquitin), superpromoter activity was approximately equivalent to those of the other promoters in both maize Black Mexican Sweet suspension cells and in stably transformed maize plants. The addition of a maize ubiquitin intron downstream of the superpromoter did not enhance activity in stably transformed maize.The availability of convenient vectors harboring a strong promoter that is active in all or most cells of different plant species would be useful for a variety of applications in plant molecular biology. We previously described a novel synthetic promoter consisting of a trimer of the octopine synthase (ocs) transcriptional activating element (ocs activator) linked to the mannopine synthase2# (mas2#) activator-promoter region (Ni et al., 1995). Initial studies in tobacco (Nicotiana tabacum) indicated that this promoter, called the superpromoter, could direct expression of GUS activity to a level 2-to 20-fold higher than the commonly used enhanced double cauliflower mosaic virus (CaMV) 35S promoter (Ni et al., 1995). The activity of the superpromoter was highest in roots, but also was high in leaves and stems.The superpromoter was originally created by ligating three ocs activator fragments (positions 2333 to 2116 relative to the transcription start site [Leisner and Gelvin, 1988]) from the ocs gene to the mas2# activator-promoter region (2318 to 165 relative to the transcription start site [Ellis et al., 1984]), all from the Agrobacterium tumefaciens Ti-plasmid pTiA6. The construction of the original superpromoter resulted in the repeated presence within the promoter of several commonly used restriction endonuclease sites (BamHI, EcoRI, HindIII), as well as the presence of the restriction endonuclease sites PstI and XhoI. This feature precluded easily linking genes to the promoter. In addition, it complicated further analysis of T-DNA insertions in the plant genome. We therefore modified the superpromoter, eliminating most of these internal restriction endonuclease sites. This modified superpromoter (MSP) formed the basis for the construction of several novel plant expression and T-DNA binary vectors. Here, we describe these vectors...