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Abstract: Using laying hens, we investigated whether position of a nest box, both within the pen and relative to other nest boxes, influenced the preference for a nest box, and how a sudden and marked change to the preferred box influenced the use of nest boxes by the hens. Groups (n =12) of 15 Isa Warren hens were housed in pens, each with five identical nest boxes in different positions: Two single (in a corner or not) and a triplet of nest boxes (one of which in a corner). The use of nest boxes was determined by the number of eggs laid daily in each box. Three experiments, each lasting 10 days, were carried out. First, the undisturbed use of each of the nest box types was investigated, and a strong preference (P <0.001) was found for single nest boxes in a corner, with 62% of the nest box eggs laid there. Second, each of the hen groups was moved to another pen allocated at random, and where the configuration of nest box types differed from that of their original pen. An effect of nest box type was found (P <0.001), with 41% of all eggs laid in the single nest box in a corner, and 26% of eggs laid in the corner triplet nest box. Third, the attractiveness of the preferred nest box within each pen was reduced by removing the Astroturf mat from the nest box floor, exposing the wire netting below. This resulted in a change of nest box use (P <0.001) from the single, corner nest box (67%; n =6) to the corner triplet nest box (37%) and the single nest box not in a corner (35%), and from the corner triplet nest box (48%; n =5) to the remaining triplet nest boxes (40% and 22%, respectively). The initial preference for a single nest box in a corner was probably due to a combination of isolation and view of the surroundings provided by this type of nest box. The manipulations in experiments II and III revealed that some hens were location conservative, i.e. continued laying in a corner location (or as close to that as possible), whereas others were isolation conservative, i.e. continued laying in the most isolated nest box despite it being positioned in a different area of the pen. [Copyright &y& Elsevier]