The term "functional connectivity" is used to denote correlations in activation among spatially-distinct brain regions, either in a resting state or when processing external stimuli. Functional connectivity has been extensively evaluated with several functional neuroimaging methods, particularly PET and fMRI. Yet these relationships have been quantified using very different measures and the extent to which they index the same constructs is unclear. We have implemented a variety of these functional connectivity measures in a new freely available MATLAB toolbox. These measures are categorized into two groups: whole time-series and trial-based approaches. We evaluate these measures via simulations with different patterns of functional connectivity and provide recommendations for their use. We also apply these measures to a previously published fMRI data set (Siegle, G.J., Thompson, W., Carter, C.S., Steinhauer, S.R., Thase, M.E., 2007. Increased amygdala and decreased dorsolateral prefrontal BOLD responses in unipolar depression: related and independent features. Biol. Psychiatry 610 (2), 198-209) in which activity in dorsal anterior cingulate cortex (dACC) and dorsolateral prefrontal cortex (DLPFC) was evaluated in 32 control subjects during a digit sorting task. Though all implemented measures demonstrate functional connectivity between dACC and DLPFC activity during event-related tasks, different participants appeared to display qualitatively different relationships.