The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three groups in terms of proliferation rate or expression of anti-apoptotic proteins. However, cyclin D1+ cases were always well differentiated and were more likely to show a lymphoplasmocytoid differentiation (chi-square = 9.55). Overall, constitutive activation of Stat 3 was found in almost half (48%) of the investigated MM cases. However, this does not seem to have a major impact on the expression of anti-apoptotic proteins and proliferation. We showed that cyclin D1 overexpression and Stat 3 activation are, mutually exclusive events in MM (P = 0.0066). The universal expression of Mcl-1, independent of activated Stat 3, suggests that its expression is constitutive and that it might play an important role in the pathogenesis of MM.