We present the first measurements of relative velocity statistics of inertial particles in a homogeneous isotropic turbulent flow with three-dimensional holographic particle image velocimetry (holographic PIV). From the measurements we are able to obtain the radial relative velocity probability density function (PDF) conditioned on the interparticle separation distance, for distances on the order of the Kolmogorov length scale. Together with measurements of the three-dimensional radial distribution function (RDF) in our turbulence chamber, these statistics, in principle, can be used to determine interparticle collision rates via the formula derived by Sundaram and Collins (1997). In addition, we show temporal development of the RDF, which reveals the existence of an extended quasi-steady-state regime in our facility. Over this regime the measured two-particle statistics are compared to direct numerical simulations (DNS) with encouraging qualitative agreement. Statistics at the same Reynolds number but different Stokes numbers demonstrate the ability of the experiment to correctly capture the trends associated with particles of different inertia. Our results further indicate that even at moderate Stokes numbers turbulence may enhance collision rates significantly. Such experimental investigations may prove valuable in validating, guiding and refining numerical models of particle dynamics in turbulent flows. (C) 2009 Elsevier Ltd. All rights reserved.