We have developed an automated method for discovering tissue‐specific regulation of alternative splicing through a genome‐wide analysis of expressed sequence tags (ESTs). Using this approach, we have identified 667 tissue‐specific alternative splice forms of human genes. We validated our muscle‐specific and brain‐specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue‐specific alternative splice forms is highest in brain, while eye_retina, muscle, skin, testis and lymph have the greatest enrichment of tissue‐specific splicing. Overall, 10–30% of human alternatively spliced genes in our data show evidence of tissue‐specific splice forms. Seventy‐eight percent of our tissue‐specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue‐specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney‐specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N‐terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue‐specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues.