Agroinoculation is a technique permitting the transmission of geminivirus genomes cloned in Agrobacterium tumefaciens into a wide variety of mono- and dicotyledonous host plants. Most geminiviruses are obligately transmitted by insect vector species under natural conditions; therefore, agroinoculation has greatly simplified the study of this group of viruses. In many cases, agroinoculation has replaced insect transmission, and has been used to compare virulence characteristics among viruses. Here we report on the discovery that, in agroinfectious Maize streak virus constructs, the orientation of cloned viral genomes relative to the Cauliflower mosaic virus 35S (CaMV35S) promoter of the binary cloning vector pBI121 can significantly affect agroinfectivity of the constructs. Rates at which plants became symptomatic were significantly higher when agroinoculating maize seedlings with constructs containing the CaMV35S promoter upstream of the viral replication-associated protein (Rep) gene than when the same viruses were cloned either in the opposite orientation or into a vector without a strong eukaryotic promoter sequence. Plants infected using the construct with Rep cloned downstream of the CaMV35S promoter also displayed more stunting and, in the early stages of the infection, more severe chlorotic streak symptoms.