Recently, Volk, Geiger, and Raz (Cancer Res., 44: 811-824, 1984) addressed the question of whether variations in actin organization in clones of the murine K-1735 melanoma tumor correlated with their metastatic capability. Using immunofluorescence techniques, they found that clones which had a more ordered actin network were less metastatic, whereas clones having a diffuse actin staining pattern were more metastatic. Similarly, we have found that in the Dunning rat R3327 prostatic adenocarcinoma tumor system, the non-metastatic (less than 0.1%) H-prostatic tumor cell line has a prominent network of actin filament bundles, whereas the highly metastatic (greater than 90%) MatLyLu cell line has a diffuse actin staining pattern. In the low-metastatic (less than 10%) AT1 cell line an intermediate actin organization between H and MatLyLu was observed. Analysis of cell extracts from H- and MatLyLu-cells revealed differences in the level of activity of cellular proteins which affect actin filament assembly and structure in a manner similar to that of the cytochalasins, fungal metabolites which bind with high affinity to the fast-growing end of actin filaments. Extracts of MatLyLu were significantly more effective than those of H-cells in decreasing the extent of actin filament network formation and in inhibiting the rate of filament assembly by blocking monomer addition onto the fast-growing end. Measurements of spin-lattice nuclear magnetic resonance water proton relaxation times (T1) were made in surgically removed tumor tissue from four sublines (H, AT1, MatLyLu, and MatLu) of the Dunning R3327 tumor system. The highly metastatic cell lines had significantly longer water proton T1 relaxation times than did the lines with low metastatic potential. These differences in T1 may reflect the observed alterations in organization of actin filaments within these various sublines of the Dunning R3327 prostatic adenocarcinoma tumor system.