To examine the effects of ligand engagement and accessory molecule juxtaposition on T cell receptor (TCR) signaling, we prepared LFA-3/ICAM-1 Rg and LFA-3/VCAM-1 Rg bispecific immunoglobulin fusion proteins (Rg, recombinant globulin). These novel fusion proteins allowed us to examine the effects of ligand driven co-engagement of T cell proteins CD2 and LFA-1 or CD2 and VLA-4 on TCR-dependent mobilization of intracellular Ca2+. We observed that preincubation of resting T cells with LFA-3/ICAM-1 Rg or LFA-3/VCAM-1 Rg fusion proteins resulted in significantly enhanced mobilization of intracellular Ca2+ following TCR-accessory molecule cross-linking relative to T cells preincubated with each of the monospecific Rgs alone or with combinations of the monospecific Rg fusion proteins. In addition, such coengagement stimulated TCR-dependent activation and tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1). These results suggest that when T cells interact with antigen presenting cells the engagement of multiple cell adhesion molecules such as CD2, LFA-1, and VLA-4 primes the T cell to respond more effectively to signals delivered through the TCR.