Numerous studies have shown the merits of targeting the costs of conservation besides environmental benefits and aligning payments for ecosystem services with incurred costs. However, cost-effective and precise estimation of site specific opportunity costs is a major challenge. In this paper we test two approaches to estimate opportunity costs of conservation: One approach derives opportunity costs from annual land rents, and the other models regresses opportunity costs on easily obtainable and difficult to manipulate spatial and socio-economic independent variables such as soil quality. None of these approaches appeared to estimate opportunity costs sufficiently well. But since this judgment is based on how well the estimates compare to the reference opportunity costs, which were computed from farm budgets, we also considered potential flaws in the reference data and tested their plausibility. The tests confirmed the plausibility of data. Based on the results presented in this paper none of the two cost estimation approaches can be recommended for practical application in conservation programs. Yet, further research is necessary to confirm these findings giving special attention to the techniques that are applied to deliver reference point data on opportunity costs.