Previous studies have shown that over time in culture opossum kidney (OK) cells are endowed with increased Na(+),K(+)-ATPase activity and expression (Silva et al., 2006, J Membr Biol 212:163-175; Silva and Soares-da-Silva, 2007, Am J Physiol Regul Integr Comp Physiol 293:R1764-R1770). The present work evaluated the cytoskeleton reorganization in OK cells at passages 40 and 80 in culture and its possible relationship with membrane transport proteins and cell morphology. It is shown that OK cells with 80 passages in culture have increased size, internal complexity, and total protein expression. In OK cells with 80 passages in culture the use of in-cell western showed that ezrin/radixin/moesin complex was increased by 20%. The most abundant ankyrin-G isoform in OK cells with 40 passages was the approximately 200/220 kDa isoform, whereas in OK cells with 80 passages the most abundant isoform was the approximately 170 kDa isoform. The spectrin-betaII approximately 240 kDa isoform, the predominant isoform in OK cells with 40 passages, was marginally detected in OK cells with 80 passages. Besides Na(+),K(+)-ATPase, GLUT2, and NHE3 expression was also significantly increased in OK cells with 80 passages. It is concluded that the prolonged cell passaging of OK cells results in an interesting and valuable experimental model to analyze the reorganization of the renal cell cytoskeleton proteins and its relationship with transporter and signaling membrane proteins.