Arg8-vasopressin (AVP), a circadian clock-controlled gene product, is released from the hypothalamic suprachiasmatic nuclei (SCN) in mice in a circadian fashion. Previously reported differences in two mouse lines, initially selected for thermoregulatory nest-building behavior (building small nests (S-mice) or big nests (B-mice)) with different circadian organization of behavior and in number of SCN-AVP immunoreactive neurons, were further investigated. We confirmed and expanded the finding that S-mice exhibited constant high levels of SCN-AVP content with no apparent circadian rhythmicity, whereas B-mice had lower numbers of AVP positive cells which varied with time of day. We found that AVP mRNA expression levels at midnight and midday were similar in both lines, as established by in situ hybridization. When AVP transport and release were blocked by colchicine, SCN-AVP immunoreactivity was similar in both lines. This suggests that differences in SCN-AVP content depend on transport or release. Organotypic SCN cultures of B-mice showed more AVP release per neuron than cultures of S-mice. These results reveal that on a mechanistic level the mouse lines differed in transport and/or release of AVP in the SCN, rather than differential regulation of AVP gene transcription or number of AVP immunoreactive neurons.