Purebred laying hen lines of White Leghorn (WL) origin have been found to be more flighty and to show more feather pecking than lines of Rhode Island Red (RIR) origin. It has been found, however, that when RIR birds were housed together with WL birds, RIR birds became more flighty and those mixed groups developed more feather damage than pure-line cage-housed groups. It is unknown, however, whether this effect of social environment is accompanied by changes in stress-related behavior and neurophysiological activity, which are assumed to be associated with increased feather damage. The objective of this study was therefore to investigate the effects of genetic origin (WL or RIR) and social environment (mixed or pure groups) on behavioral response to manual restraint and monoamine functioning. Monoamine functioning was measured by brain serotonin (5-HT) and dopamine turnover. Furthermore, correlations between 5-HT turnover in the brain and peripheral measures of 5-HT in the blood were calculated. Experimental birds, housed either with other birds from the same genetic origin (pure groups) or with both RIR and WL birds (mixed groups) from hatching onward, were subjected to a manual restraint test at 47 wk of age. The WL birds struggled less during restraint and had higher dopamine and 5-HT turnover levels after restraint than did RIR birds. The WL birds also showed higher levels of platelet 5-HT uptake than did RIR birds. No effects of social environment were found. Blood and brain 5-HT measures were found to be correlated, with correlations ranging from 0.34 to 0.57, which seems to offer opportunities for less invasive peripheral indicators of 5-HT activity. In conclusion, genetic origin, but not social environment, affected the behavioral response to manual restraint and monoamine functioning in laying hens.