TULA belongs to a two‐member family: TULA (STS‐2) is a lymphoid protein, whereas STS‐1/TULA‐2 is expressed ubiquitously. TULA proteins were implicated in the regulation of signaling mediated by protein tyrosine kinases (PTKs). The initial experiments did not fully reveal the molecular mechanism of these effects, but suggested that both TULA proteins act in a similar fashion. It was shown recently that STS‐1/TULA‐2 dephosphorylates PTKs. In this study, we analyzed the effects of TULA proteins on Syk, a PTK playing an important role in lymphoid signaling. First, we have shown that TULA‐2 decreases tyrosine phosphorylation of Syk in vivo and in vitro and that the intact phosphatase domain of TULA‐2 is essential for this effect. We have also shown that TULA‐2 exhibits a certain degree of substrate specificity. Our results also indicate that inactivated TULA‐2 increases tyrosine phosphorylation of Syk in cells co‐transfected to overexpress these proteins, thus acting as a dominant‐negative form that suppresses dephosphorylation of Syk caused by endogenous TULA‐2. Furthermore, we have demonstrated that phosphatase activity of TULA is negligible as compared to that of TULA‐2 and that this finding correlates with an increase in Syk tyrosine phosphorylation in cells overexpressing TULA. This result is consistent with the dominant‐negative effect of inactivated TULA‐2, arguing that TULA acts in this system as a negative regulator of TULA‐2‐dependent dephosphorylation. To summarize, our findings indicate that TULA proteins may exert opposite effects on PTK‐mediated signaling and suggest that a regulatory mechanism based on this feature may exist. J. Cell. Biochem. 104: 953–964, 2008. © 2008 Wiley‐Liss, Inc.