AbstractThe hypothalamic suprachiasmatic (SCN) clock contains several neurochemically defined cell groups that contribute to the genesis of circadian rhythms. Using cell-specific and genetically targeted approaches we have confirmed an indispensable role for vasoactive intestinal polypeptide-expressing SCN (SCN^VIP) neurons, including their molecular clock, in generating the mammalian locomotor activity (LMA) circadian rhythm. Optogenetic-assisted circuit mapping revealed functional, di-synaptic connectivity between SCN^VIP neurons and dorsomedial hypothalamic neurons, providing a circuit substrate by which SCN^VIP neurons may regulate LMA rhythms. In vivo photometry revealed that while SCN^VIP neurons are acutely responsive to light, their activity is otherwise behavioral state invariant. Single-nuclei RNA-sequencing revealed that SCN^VIP neurons comprise two transcriptionally distinct subtypes, including putative pacemaker and non-pacemaker populations. Altogether, our work establishes necessity of SCN^VIP neurons for the LMA circadian rhythm, elucidates organization of circadian outflow from and modulatory input to SCN^VIP cells, and demonstrates a subpopulation-level molecular heterogeneity that suggests distinct functions for specific SCN^VIP subtypes.