Chloroplasts are a key feature of most guard cells; however, the function of these organelles in stomatal responses has been a subject of debate. This review examines evidence for and against a role of guard cell chloroplasts in stimulating stomatal opening. Controversy remains over the extent to which guard cell Calvin cycle activity contributes to stomatal regulation. However, this is only one of four possible functions of guard cell chloroplasts; other roles include supply of ATP, blue-light signalling and starch storage. Evidence exists for all these mechanisms, but is highly dependent upon species and growth/measurement conditions, with inconsistencies between different laboratories reported. Significant plasticity and extreme flexibility in guard cell osmoregulatory, signalling and sensory pathways may be one explanation. The use of chlorophyll a fluorescence analysis of individual guard cells is discussed in assessing guard and mesophyll cell physiology in relation to stomatal function. Developments in transgenic and molecular techniques have recently provided interesting, albeit contrasting, data regarding the role of these highly conserved organelles in stomatal function. Recent studies examining the link between mesophyll photosynthesis and stomatal conductance are discussed. An enhanced understanding of these processes may be fundamental in generating crop plants with greater water use efficiencies, capable of combating future climatic changes.