Calcium imaging techniques were used to study intracellular free calcium ion regulation in isolated Müller cells in response to changes in extracellular potassium concentration and to caffeine and ryanodine. Müller cells were dissociated from the adult tiger salamander (Ambystoma tigrinum) retina and studied using the calcium indicator Fura‐2 and video imaging microscopy techniques. Our results demonstrate that elevation of extracellular potassium in the presence of extracellular calcium evokes an increase in intracellular calcium ([Ca2+]i) throughout the length of the Müller cell. In contrast, in the absence of extracellular calcium, elevation of extracellular potassium can trigger a long latency, wave‐like increase in [Ca2+]i that begins in the apical region of the Müller cell and moves toward the endfoot. A similar calcium wave can be evoked in Müller cells when they are exposed to caffeine or ryanodine, agents that cause release of calcium from intracellular stores in many cell types. These data suggest that [Ca2+]i may be altered in Müller cells through an extracellular pathway as well as through a ryanodine‐sensitive intracellular release mechanism. The functional consequences of these changes in [Ca2+]i remain to be elucidated. © 1995 Wiley‐Liss, Inc.