Continuous measurement and imaging of the intracellular free calcium ion concentration ([Ca ²⁺ ] _i ) of mitotic and interphase PtK ₁ cells was accomplished with the new fluorescent Ca ²⁺ indicator fura-2. No statistically significant difference between basal [Ca ²⁺ ] _i of interphase and mitotic cells was detected. However, mitotic cells showed a rapid elevation of [Ca ²⁺ ] _i from basal levels of 130 n M to 500 to 800 n M at the metaphase-anaphase transition. The [Ca ²⁺ ] _i transient was brief, lasting approximately 20 seconds and the elevated [Ca ²⁺ ] _i appeared uniformly distributed over the entire spindle and central region of the cell. The close temporal association of the [Ca ²⁺ ] _i transient with the onset of anaphase suggests that calcium may have a signaling role in this event.