Dendritic spines are postsynaptic structures and they store memory. We demonstrated that sex-steroids are synthesized locally in the hippocampus in addition to the gonads (Hojo et al., 2004, 2009). Here, we focus on the rapid effect of testosterone (T) dihydrotestosterone (DHT) and estradiol (E2), on the synaptic plasticity via synaptic steroid receptors. We examined the rapid modulation of spines (= postsynapses) by application of sex-steroids, by means of single spine analysis of Lucifer-Yellow injected neurons in adult male hippocampal slices. 3D images were obtained using confocal microscope. Following a 2 h treatment with T, DHT and E2 in CA1 region, the density of spines was significantly increased by approximately 1.3 fold. Automated mathematical analysis (by Spiso-3D software) of spines clarified different effects of these three sex-steroids (Mukai et al., 2011). DHT increased large-head and middle-head spines, T increased large-head and small-head spines, but E2 increased small-head spines. To determine the pathway of sex-steroid signaling, we applied selective kinase inhibitor, including the inhibitor of MAP kinase, PKA, PKC and PI3K. The inhibitor individually suppressed the increase of spines by DHT, T and E2. These results indicate that androgen and estrogen rapidly drive the multiple kinase pathways, by inducing phosphorylation of actin binding proteins. Synaptic AR and ER receptors participate in the signaling. These results show new molecular mechanisms of sex-steroid-induced neurotrophic effects on synapses.