Effects of pamamycins on intracellular calcium concentration ([Ca2+]i) in Streptomyces alboniger were examined by microscopic fluorometry with Fura-2 as the indicator. Pamamycin-607, an aerial mycelium-inducing substance isolated from S. alboniger IFO 12738, induced a transient increase in [Ca2+]i dose-dependently. At 20 μM, [Ca2+]i increased immediately after treatment, peaked at 2.0 times the baseline, and gradually decreased; the response magnitude diminished with lower concentrations (10, 5 μM). Digitized images showed the [Ca2+]i increase starting from the inner edge of the mycelium and spreading outward. The inactive homologue pamamycin-649 had no effect on [Ca2+]i. Previous studies revealed Ca2+ regulates aerial mycelium formation in actinomycetes (including S. alboniger) and pamamycin-607 requires Ca2+ for activity. Further experiments demonstrated that mycelia treated with pamamycin-607 for 1 min formed aerial mycelia on cerulenin-containing plates (which inhibit endogenous pamamycin production), while those treated with pamamycin-649 did not. These results suggest that pamamycin-607-induced [Ca2+]i increase triggers aerial mycelium formation in S. alboniger. This is the first report of measuring [Ca2+]i in an actinomycete and correlating it with a physiological function.