In addition to its potential contribution to improving animal welfare, the study of the genetics of cattle behavior may provide more general insights into the genetic control of such complex traits. We carried out a genome scan in a Holstein x Charolais cross cattle population to identify quantitative trait loci (QTL) influencing temperament-related traits. Individuals belonging to the second-generation of this population (F(2) and backcross individuals) were subjected to 2 behavioral tests. The flight from feeder (FF) test measured the distance at which the animal moved away from an approaching human observer, whereas the social separation (SS) test categorized different activities which the animal engaged in when removed from its penmates. The entire population was genotyped with 165 microsatellite markers. A regression interval mapping analysis identified 29 regions exceeding the 5% chromosome-wide significance level, which individually explained a relatively small fraction of the phenotypic variance of the traits (from 3.8% to 8.4%). One of the significant associations influencing an FF test trait on chromosome 29 reached the 5% genome-wide significance level. Eight other QTL, all associated with an SS test trait, reached the 1% chromosome-wide significance level. The location of some QTL coincided with other previously reported temperament QTL in cattle, whereas those that are reported for the first time here may represent general loci controlling temperament differences between cattle breeds. No overlapping QTL were identified for the traits measured by the 2 different tests, supporting the hypothesis that different genetic factors influence behavioral responses to different situations.