A method for circumventing the blood-brain barrier (BBB), called convection-enhanced delivery (CED), involves directly infusing therapeutics into the brain tissue. The behavior of the therapeutics once in the tissue is not well understood. To gain insight into the behavior of materials introduced into the brain during a CED-like infusion, we used two-photon fluorescence microscopy to observe infusate movement in vivo. With our experiments, we were able to sample the infusate volume with high temporal (~30 s volume sample) and spatial (~2.5 µm3 voxel size) resolution. With observable locations of the infused materials at many time points throughout the infusion, we determined important position, velocity, and volume characteristics of each infusion. For smaller, less-rigid materials, we saw larger distribution volume relative to larger, more-rigid materials. Furthermore, we were able to directly observe materials entering and traveling in the perivascular spaces (PVS), which are thin annular regions surrounding blood vessels in the brain. PVS are believed to be important in a number of neurological disorders and neural homeostasis, and we have shown that they play an influential role in the movement of infused materials. For a given timepoint in an infusion, the average PVS occupancy of infused materials can be a large portion of the total volume observed, depending on the location of an available space relative to the infusion needle outlet. Given the large amount of infused material entering the PVS, we theorize how this knowledge can be used to influence clinical treatments of neurological disorders in order to have better control of where the infused materials will go during different parts of an infusion.