Background Psychiatric disorders such as schizophrenia are worsened by stress, and working memory deficits are often a central feature of illness. Working memory is mediated by the persistent firing of prefrontal cortical (PFC) pyramidal neurons. Stress impairs working memory via high levels of dopamine D 1 receptor (D 1 R) activation of cyclic adenosine monophosphate signaling, which reduces PFC neuronal firing. The current study examined whether D 1 R-cyclic adenosine monophosphate signaling reduces neuronal firing and impairs working memory by increasing the open state of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, which are concentrated on dendritic spines where PFC pyramidal neurons interconnect. Methods A variety of methods were employed to test this hypothesis: dual immunoelectron microscopy localized D 1 R and HCN channels, in vitro recordings tested for D 1 R actions on HCN channel current, while recordings in monkeys performing a working memory task tested for D 1 R-HCN channel interactions in vivo. Finally, cognitive assessments following intra-PFC infusions of drugs examined D 1 R-HCN channel interactions on working memory performance. Results Immunoelectron microscopy confirmed D 1 R colocalization with HCN channels near excitatory-like synapses on dendritic spines in primate PFC. Mouse PFC slice recordings demonstrated that D 1 R stimulation increased HCN channel current, while local HCN channel blockade in primate PFC protected task-related firing from D 1 R-mediated suppression. D 1 R stimulation in rat or monkey PFC impaired working memory performance, while HCN channel blockade in PFC prevented this impairment in rats exposed to either stress or D 1 R stimulation. Conclusions These findings suggest that D 1 R stimulation or stress weakens PFC function via opening of HCN channels at network synapses.