Conditional gene targeting using the Cre/loxP strategy has proven to be very useful for studies of glucose homeostasis, tissue function and dysfunction in diabetes, and pancreas development. However, use of this strategy over the past decade has revealed a variety of experimental caveats, many of which are a direct consequence of the procedures used to generate Cre-driver lines. We discuss frequently encountered experimental artefacts, the advantages of using bacterial artificial chromosome-derived transgenes or performing a Cre knockin for improving the specificity of expression, and systems for regulating Cre activity. In addition, recent studies indicate that high amounts of Cre in the pancreatic beta-cell may cause glucose intolerance and impaired insulin secretion. However, these findings, while serving as a reminder for simple experimental controls, are unlikely to diminish utilization of this very powerful and useful technology.