Recent evidence that insulin receptors are concentrated in the hypothalamus and olfactory bulb suggests that insulin may have an important regulatory function in these regions. This hypothesis would be supported by finding that insulin itself is concentrated in the hypothalamus and olfactory bulb. Therefore, we extracted the hypothalamus and olfactory bulb, as well as the amygdala, hippocampus, cerebral cortex, hindbrain, midbrain, and whole brains, of fasted male Wistar rats and measured immunoreactive insulin (IRI). Recovery of insulin added to extraction volumes of 500-1000 microliters was 90-100%, whereas recovery of insulin from tissue extracts was 63%. Mean IRI concentrations were relatively uniform throughout the brain (0.19 ng/g wet wt; uncorrected for recovery) and were significantly lower than plasma levels (1.03 ng/ml). Nevertheless, IRI concentrations were significantly higher in hypothalamus (0.39 +/- 0.02 ng/g; P less than 0.01) and olfactory bulb (0.37 +/- 0.02 ng/g; P less than 0.05) compared to those in other brain regions sampled.