The molecular–cellular architecture of the hypothalamic preoptic area is not well understood. Here, more than 1 million cells from the preoptic region of mouse hypothalamus were analysed using single-cell RNA sequencing (scRNAseq) and multiplexed error-robust fluorescence in situ hybridization (MERFISH). scRNAseq provided a detailed analysis of gene expression in individual cells, and MERFISH enabled gene expression profiling of, and imaging of activity markers in, live cells, thus providing a spatial context to the expression patterns at high resolution. Applying these techniques after various behaviours known to involve the preoptic area revealed differences in FOS expression patterns in ~70 specific neuronal populations, deepening our understanding of this complex brain area.