Interstitial cells of Cajal (ICC) generate electrical pacemaker activity in the gastrointestinal (GI) tract known as slow waves, which regulate GI motility. ICC express both the Kit receptor tyrosine kinase protein and a Ca2+-activated Cl--channel, encoded by the anoctamin1 (Ano1) protein, which is an essential contributor to the Ca2+ cycling of ICC and slow wave pacemaking. Recent dye-loading imaging studies have demonstrated Ca2+ transients in ICC in isolated tissue preparations. The main aim of this study was to develop a method that allows Ca2+ transients to be registered to structural ICC network data. Confocal image stacks of ICC labeled for Kit or Ano1 and Ca2+ recording data were processed using a thresholding protocol. The Ca2+ transients were then registered to the ICC structural network. First, a general idea of the placement was found by mapping the field-of-view of the Ca2+ transient data to the distorted tissue that contained the ICC network image. The errors in the registration were then corrected for by warping the internal Ca2+ transient field according to the structural network. In data sets from tissues with induced, targeted knockdown of Ano1 expression in a subset of ICC, agreement between the Ca2+ transient data and structural network was 68 ± 10%. This level of agreement allowed selective extraction of Ca2+ data from Ano1-positive (Ano1+) and Ano1-negative (Ano1-) ICC. In the future, this technique will allow investigation into the functional properties of ICC in relation to the level of knockdown of specific ICC associated proteins.