Rat brain cell aggregates represent a three-dimensional tissue culture system of brain tissue in the form of small, multicellular spheroids. In the present work, we have infected these 'minibrains' with neurovirulent, nonneurovirulent, and nonreplicating strains of HSV-1. The neurovirulent strains 17+ and KOS(M) spread rapidly through the aggregates, while the nonreplicating ICP4 deletion mutant KD6 infected cells only at the periphery of the aggregates. Spread and replication of the nonneurovirulent strains RE6 and tk-7, and to some extent also of R13/1, were restricted. The interaction between different strains of HSV-1 and the rat brain cell aggregates is thus comparable to that seen in the brain, suggesting that the aggregates represent a useful tool for studying HSV-1 infection of brain tissue in vitro.