The forms and stratification of soil phosphorus (P) and their relationship to mobile forms of P were investigated in soils collected from a subcatchment used for grazing of dairy cattle in the Adelaide Hills, South Australia. Phosphorus in the soils was highly stratified. The concentration of calcium chloride extractable P in the 0–0.01 m increment was, on average, 5.7 times greater than in the 0.05–0.10 m increment. Organic P (% of total P) in the top 0.01 m was significantly (P < 0.001) related to soil P content such that low P soils (total P of ~600 mg/kg) had high proportions of P_o (~65%), whereas high P soils (total P of ~2000 mg/kg) had low proportions (~25%) of P_o. Runoff P from these soils was predominantly (86%) dissolved (i.e. <0.45 μm). There was a significant (P < 0.001) exponential relationship between Olsen P in the top 0.01 m and dissolved P concentration in runoff. The form of dissolved P in runoff from soil in repacked trays was also significantly (P < 0.001) related to soil P. Runoff from low P soils (high P_o) had high proportions (>50%) of dissolved unreactive P (DUP), whereas runoff from high P soils (low P_o) had low proportions of DUP (<10%). Ultrafiltration of runoff samples revealed that 94 and 65% of the dissolved reactive P and DUP, respectively, was subcolloidal (i.e. <1 nm). These results highlight the relationship between soil fertility, the forms of soil P, and the concentrations and forms of P mobilised in runoff. Such relationships need to be considered in further studies of P mobilisation and the subsequent development of strategies designed to reduce runoff P concentrations.