AS (alternative splicing) is a post-transcriptional process which regulates gene expression through increasing protein complexity and modulating mRNA transcript levels. Regulation of AS depends on interactions between trans-acting protein factors and cis-acting signals in the pre-mRNA (precursor mRNA) transcripts, termed ‘combinatorial’ control. Dynamic changes in AS patterns reflect changes in abundance, composition and activity of splicing factors in different cell types and in response to cellular or environmental cues. Whereas the SR protein family of splicing factors is well-studied in plants, relatively little is known about other factors influencing the regulation of AS or the consequences of AS on mRNA levels and protein function. To address fundamental questions on AS in plants, we are exploiting a high-resolution RT (reverse transcription)–PCR system to analyse multiple AS events simultaneously. In the present paper, we describe the current applications and development of the AS RT–PCR panel in investigating the roles of splicing factors, cap-binding proteins and nonsense-mediated decay proteins on AS, and examining the extent of AS in genes involved in the same developmental pathway or process.