Cell adhesion is a multistep process that requires the interaction of integrins with their ligands in cell attachment, the activation of integrin-triggered signals, and cell spreading. Integrin beta subunit cytoplasmic domains (beta tails) participate in regulating each of these steps; however, it is not known whether the same or different regions within beta tails are required. We generated a panel of amino acid substitutions within the beta1 and beta3 cytoplasmic domains to determine whether distinct regions within beta3 tails regulate different steps in adhesion. We expressed these beta cytoplasmic domains in the context of interleukin 2 (IL-2) receptor (tac) chimeras and tested their ability to activate tyrosine phosphorylation, to regulate beta1 integrin conformation and to inhibit beta1 integrin function in cell attachment and spreading. We found that many of the mutant beta3 and beta3 chimeras either had no effect on these parameters or dramatically inhibited the function of the beta tail in most assays. However, one set of analogous Ala substitutions in the beta1 and beta3 tails differentially affected the ability of the tac-beta3 and tac-beta3 chimeras to activate tyrosine phosphorylation. The tac-beta1 mutant containing Ala substitutions for the VTT motif did not signal, whereas the analogous tac-beta3 mutant was able to activate tyrosine phosphorylation, albeit not to wild-type levels. We also identified a few mutations that inhibited beta tail function in only a subset of assays. Ala substitutions for the Val residue in the VTT motif of the beta1 tail or for the conserved Asp and Glu residues in the membrane-proximal region of the beta3 tail greatly diminished the ability of tac-beta1 and tac-beta3 to inhibit cell spreading, but had minimal effects in other assays. Ala substitutions for the Trp and Asp residues in the conserved WDT motif in the beta1 tail had dramatic effects on the ability of tac-beta1 to regulate integrin conformation and function in cell spreading, but had no or intermediate effects in other assays. The identification of mutations in the beta1 and beta3 tails that specifically abrogated the ability of these beta tails to regulate beta1 integrin conformation and function in cell spreading suggests that distinct protein interactions with beta tails regulate beta cytoplasmic domain function in these processes.