Several previous studies highlighted the importance of using field-collected soils instead of artificially spiked contaminated soils for phytotoxicity tests. However, the use of field-collected soils presents several difficulties for interpretation of results, due to presence of various contaminants in the soil and unavoidable differences in the physicochemical properties of the tested soils. The objective of this study was to estimate thresholds of copper phytotoxicity in topsoils of 27 agricultural areas historically contaminated by mining activities in Chile. We performed standard emergence and early growth (21 days) tests (OECD 208 and ISO 11269-2) with lettuce. The response of lettuce was best explained by Cu toxicity and P deficiency. Growth of lettuce was related to soil total Cu concentration and Olsen-P and was not affected by soluble Cu (extractable by 0.1 M KNO3) or Cu2+ free ion activity of the soil solution. Thus, lettuce has a limited applicability for metal toxicity assessment in metalcontaminated soils, due to sensitivity of its response to P deficiency. However, it was possible to determine toxic thresholds for shoot concentrations of Cu in lettuce for responses of shoot and root length, suggesting that shoot concentrations of Cu in lettuce can be useful as indicators of Cu toxicity even in soils with a wide range of nutrient concentrations.