The single-stranded DNA geminiviruses produce transcripts from both strands (virion- and complementary-sense) of a nuclear double-stranded DNA molecule. In maize streak virus (MSV)-infected maize plants, approximately 80% of the complementary-sense transcripts produce the C1 protein, whilst the remaining 20% are spliced to remove a 92 nt intron and produce a C1:C2 fusion protein (Rep). Disruption of the complementary-sense 3' splice site abolished virus replication. The majority of the virion-sense transcripts initiated one nucleotide upstream of the V1 (movement protein) gene and a minority a further 141 nucleotides upstream. A 76 nt intron, with features typical of plant introns, was identified within the V1 gene, upstream of the coat protein gene. Spliced and unspliced forms of each virion-sense transcript were produced, but they differed in splicing efficiency. Approximately 50% of the major transcript and less than 10% of the minor transcript were processed. Mutagenesis of the consensus 5' splice site in the V1 gene resulted in the use of alternative cryptic splice sites, confirming the importance of splicing for MSV infection. Spliced virion-sense transcripts were also identified in tissue infected with the closely-related Digitaria streak virus (DSV) but not with another subgroup I geminivirus, wheat dwarf virus. Collectively, the multiple transcript initiation sites and different splicing efficiencies suggest that splicing is an important feature in the regulation of both early and late gene expression in MSV and DSV.