Clinical and basic studies demonstrate that estrogen (E-sub-2)-based therapies influence anxiety and mood, but the receptor targets (e.g., a or ss isoform of the estrogen receptor, ER) for these effects requires further investigation. To address the specificity of E2's anxiolytic-like effects through ERss, anxiety, motor, and nociceptive behavior of ovariectomized, wildtype (WT), and ERss knockout (ssERKO) mice was examined. Mice were administered oil vehicle or ER agonists, 17ss-E2 (E2; 0.1 mg/kg; similar affinity for ERa and ERss), and a selective ER modulator, diarylpropionitrile (DPN; 0.1 mg/kg; greater affinity for ERss than ERa). Performance of mice in anxiety (open field, elevated plus maze, elevated zero maze, social interaction), motor activity (activity monitor) and nociception (tailflick, pawlick) measures was compared. Results supported our hypothesis that ERss is important in modulation of anxiety-like behavior by E2 in some tasks. Administration of E2 or DPN to WT, but not ssERKO, mice increased open field central entries, plus maze open arm time, zero maze open quadrant time, and social interaction. This pattern was neither seen in motor activity nor pain threshold measures. Thus, actions of ERss may be important for modulating anxiety-like behavior of mice.