Several bladder cancer culture systems have been developed in recent years. However, reports about successful primary cultures of superficial urothelial carcinomas (UC) are sparse. Based on the specific growth requirements of UC described previously, we developed a new and reliable culture system for superficial low-grade UC. Between November 2002 and April 2006, 64 primary cultures of bladder cancer specimens were performed. After incubating the specimens overnight in 0.1% ethylenediaminetetraacetic acid solution, tumour cells could easily be separated from the submucosal tissue. Subsequently, cells were seeded in a low-calcium culture medium supplemented with 1% serum, growth factors, non-essential amino acids and glycine. The malignant origin of the cultured cells was demonstrated by spectral karyotyping. Overall culture success rate leading to a homogenous tumour cell population without fibroblast contamination was 63%. Culture success could be remarkably enhanced by the addition of glycine to the culture medium. Interestingly, 86.4% of pTa tumours were cultured successfully compared to only 50% of the pT1 and 38% of advanced stage tumours, respectively. G1 and G2 tumours grew significantly better than G3 tumours (86, 73 and 41%, respectively). Up to three passages of low-grade UC primary cultures were possible. We describe a new and reliable culture system, which is highly successful for primary culture and passage of low-grade UC of the bladder. Therefore, this culture system can widely be used for functional experiments on early stage bladder cancer.