Abstract Replacement cost refers to the loss incurred if the ideal set of conservation areas cannot be protected due to compulsory inclusion or exclusion of some area candidates. This cost can be defined either in terms of loss of conservation value or in terms of extra acquisition cost, and it has a clear mathematical definition as a difference between the value of the unconstrained optimal solution and a constrained suboptimal solution. In this work we for the first time show how replacement cost can be calculated in the context of sequential reserve selection, where a reserve network is developed over a longer time period and ongoing habitat loss influences retention and availability of sites. In case of site exclusion, a question that can be asked is, “if a site belonging to the ideal (optimal) solution cannot be obtained, what expected loss in reserve network value does this entail by the end of the planning period given that the rest of the solution is re-organized in the most advantageous manner?” Heuristically, the proposed method achieves the ambit of combining irreplaceability and vulnerability into one score of site importance. We applied replacement cost analysis to conservation prioritization for wood-inhabiting fungi in Norway, identifying factors that influence replacement cost and urgency of site acquisition. Among other things we find that the reliability of loss rate information is important, because the optimal site acquisition order may be strongly influenced by underestimated loss rates.