Dendritic spines are postsynaptic specializations thought to regulate the strength of synaptic transmission and play a critical role in neuronal plasticity. While changes in dendritic spine density can be pharmacologically- or environmentally-induced, the widespread utility of this important measure of synaptic plasticity in vivo has been hampered by the labor-intensive nature, and potential for bias and inconsistency inherent in manual spine counting. Here we report a method for obtaining high-resolution, three-dimensional confocal images of accumbens spiny neurons labeled with a diolistically delivered lipophilic fluorescence dye (DiI) that permits automated analysis of spine density and spine head diameter. The automated quantification was verified by manual counts of spine density and electron microscopic measures of spine head diameter. The density of spines was relatively constant over 2nd to 4th order dendrites within a neuron, and spine density was normally distributed. The mean spine density (2.68 spines/microm; N = 45 neurons) was higher than previous reports, due in part to analysis in three rather than two dimensions and the capacity of lipophilic dyes to fill very thin spines. The distribution of spine head diameters was continuous and skewed to the right (mean = 0.43 microm; N = 8,891), and approximately 25% of all spines were thin and filopodia-like (< or = 0.20 microm diameter). The density of spines was not correlated with average spine head diameter or with the number of filopodia-like spines. The capacity to rapidly assess spine density and spine head diameter will facilitate quantifying spine plasticity induced by pharmacological and environmental manipulations.