OBJECTIVE To develop a technique for isolation and culture of canine insulinoma cells and assess expression of cellular hexokinases (glucokinase and hexokinase I) and expression and secretion of insulin from these cells in vitro. SAMPLE Pancreatic insulinomas and normal pancreatic tissue from 4 and 3 dogs, respectively. PROCEDURES Tissues were collected by surgical excision or at necropsy. Insulinoma cells from 2 dogs were cultured for up to 10 weeks with standard techniques; insulin synthesis in vitro was confirmed by immunohistochemical analysis of freshly prepared slides of cultured cells, and insulin secretion was assessed by measurement of insulin concentrations in culture medium with an ultrasensitive mouse insulin ELISA. Expression of cellular hexokinases in insulinomas and adjacent normal (nontumor) pancreatic tissue from the same dog (n = 3) was examined by quantitative reverse transcriptase PCR assay. RESULTS Insulinoma cells survived for up to 10 weeks but did not proliferate in culture. Insulin was detected in isolated cells and secreted into culture medium for up to 10 weeks. Both cellular hexokinases were expressed; glucokinase appeared to be overexpressed in insulinomas, compared with normal pancreatic tissue from the same dogs. CONCLUSIONS AND CLINICAL RELEVANCE Canine insulinomas expressed hexokinases responsible for glucose responsiveness. Insulinoma cells were successfully maintained in short-term culture; cultured cells remained functional for 10 weeks as evidenced by cellular insulin content and had detectable secretion of insulin into the culture medium for ≥ 5 weeks. Apparent glucokinase overexpression by insulinomas suggested a possible mechanism underlying excessive insulin release by these tumors.