Primary isolates of feline immunodeficiency virus (FIV) appear to require binding to CD134 in conjunction with CXCR4(X4) to infect IL-2 dependent T-cell derived cells in culture. However, much less is known about the role of X4 for infection of cells in vivo. To investigate the correlation between X4 expression and FIV infection in cats acutely infected with FIV-C-Pgmr we used high speed fluorescence activated cell sorting and realtime PCR to co-analyze cell phenotypes from lymph node, thymus, bone marrow and blood for FIV infection and X4 expression. X4 expression was greatest in lymph node, both in frequency and in mean fluorescence intensity. The thymus demonstrated a higher proviral burden in X4+ thymic T cells (~14% in X4+ thymic T cells and 7% in X4− cells) whereas, proviral loads were similar between X4+ and X4− cell populations in all other tissues examined. Assuming a minimum of one proviral copy per cell, a maximum of ~50% of FIVpositive cells were X4+. The highest fraction of FIV-infected X4− cells was present in bone marrow. Regardless of X4 status, proviral loads were higher in lymph node and blood T cells than in B cells. These studies provide both a positive association between X4 expression and FIV infection and introduce the probability that X4− independent infection occurs in other target cells in vivo.