Interest in understanding the etiology and developing new treatments for anxiety disorders in children and adolescents has led to recent studies of neurotransmitters not traditionally associated with neural pathways for fear and anxiety. The binding of the neurotransmitter substance P (SP) to its neurokinin-1 (NK1) receptor may be a crucial component in mediating the anxiety response. While previous studies using rodent models have documented the anxiolytic effects of SP antagonists, the role of individual differences in affective temperament has not yet been examined in studies of drug response. This study used intracerebroventricular injections of the NK1 antagonist Spantide II at concentrations of 10 and 100 pmol to examine the consequences of blocking the SP-NK1 pathway in high and low line rats selectively bred for high or low levels of ultrasonic distress calls after a brief maternal separation. Affective temperament was a significant factor in determining drug response. Spantide II resulted in a significant reduction of distress calls in subjects in the high anxiety line, while low line subjects with low anxiety were resistant to the drug. These data indicate that the SP-NK1 pathway could be an important therapeutic target for the treatment of various stress disorders, but drug response might be influenced by the individual’s state anxiety or history of chronic stress.