Ionized calcium [Ca2+] in the amphibian Rana pipiens was measured over 18 mo. An annual variation about the mean of 1.10 mmol/l was observed that peaked in April (1.36 mmol/l) and attained minimal levels in October (0.84 mmol/l). Plasma total calcium [Ca]t, plasma proteins, and the bound calcium fraction also exhibited annual oscillations. The annual mean binding capacity of plasma proteins for calcium was established at 0.0175 mmol/g. Of the plasma total calcium in spring animals, nonprotein-bound calcium complexes [Ca]c were less than 3% of the total whereas 80% was composed of [Ca2+] and 17% as protein-bound calcium [Ca]pb. In autumn [Ca2+] composed 40%, [Ca]pb 30%, and [Ca]c 30% of plasma total calcium. An equilibrium exists between [Ca2+] and [Ca]c, which maintains a combined annual mean of 1.48 mmol/l calcium. Diurnal variations superimposed on the annual cycles are modulated by apparent movement of plasma water and an intrinsic 12-h rhythm for [Ca2+]. No definite associations could be made with the entrainment of specific rhythms of the three calcium fractions with the light-dark cycle.