Rat brain biopterin, the hydroxylase cofactor, was observed to distribute equally across regional subcellular fractions, rather than to codistribute neuronally with tyrosine and tryptophan hydroxylases for which it functions. Over a 24 h period with light/dark phasing, which some groups have shown to result in cycling of biopterin levels in striate and certain other regions, only the biopterin associated with the crude nuclear fraction of the striate (not associated with neurotransmitter synthesis) demonstrated a diurnal cycle. The selectivity of this perturbation response to the striate nuclear fraction suggests that (1) multiple subcellular loci of biopterin might exist independently in rat brain neurons and (2) the pterin's availability for neurotransmitter biosynthesis is limited beyond its apparent regional concentration. The demonstration of multiple independent sources of neuronal biopterin may be relevant to understanding why regional levels have been so resistant to efforts at pharmacological manipulation (only amphetamine and CRF have changed striate biopterin levels). It also shows that changes in regional hydroxylase cofactor levels may not be related to neurotransmitter synthesis, but instead may result from another presently unknown demand for the cofactor at a disparate neuronal site.