In order to reduce side effects of survivin-inhibiting anticancer therapies, we determined the expression of the survivin transcripts survivin-wild-type (survivin-wt), survivin-ΔEx3 (ΔEx3) and survivin-2B (2B) in cryo-preserved tumor and non-malignant bladder tissues (18 tumor and 22 nonmalignant samples, including 17 autologous tissue pairs) by quantitative PCR. Furthermore, we investigated the biological effects following specific inhibition of the alternative transcripts ΔEx3 and 2B in bladder cancer (BCa) cells. In BCa and non-malignant bladder tissues survivin-wt was the quantitatively dominant transcript followed by ΔEx3 and 2B. The mean mRNA expression of ΔEx3 (0.37 vs. 0.06 zmol/amol GAPDH, respectively) and 2B (0.13 vs. 0.01 zmol/amol GAPDH, respectively) was significantly higher in BCa compared to non-malignant bladder tissues, indicating their accessibility for an expression inhibition in BCa cells. Effective and long-lasting small interfering RNA-mediated inhibition of one alternative survivin transcript caused lower cell growth reduction effects (apoptosis induction, cell cycle arrest, colony formation) compared to simultaneous inhibition of multiple survivin transcripts including survivin-wt. Inhibition of one alternative survivin transcript increased the apoptosis rate by 11% vs. 33-46% when reducing several survivin transcripts. We observed no G2/M arrest or reduction of cell colony formation after inhibiting one alternative survivin transcript. Reduction of cell viability by the chemotherapeutics cisplatin, mitomycin C or gemcitabine was stronger in combination with inhibition of several survivin transcripts than in combination with the reduction of one alternative survivin splice variant. Furthermore, reducing one alternative transcript caused chemosensitization to only one chemotherapeutic agent in contrast to inhibition of several survivin transcripts. Therefore, the alternative survivin transcripts ΔEx3 and 2B do not represent reasonable targets for anticancer, at least BCa, treatment.