The actin-containing microfilaments, microtubules, and fibronectin expression of Shionogi 115 mouse mammary tumor cells were visualized by indirect immunofluorescence microscopy. Also studied was the focal adhesion distribution as revealed by interference reflection microscopy and the ability of the cells to grow in suspension culture. All these parameters were documented for androgen-responsive and -unresponsive cells grown in culture, as well as the transition of androgen-responsive to -unresponsive cells when deprived of androgen. The androgen-unresponsive cells had extensive and prominent microfilament bundles together with focal adhesions on the lower cell surface and also showed strict anchorage dependence for growth. In contrast, microfilament bundles and focal adhesions were absent from androgen-responsive cells, which furthermore had the ability to grow in suspension culture. Differences were also apparent in fibronectin expression, the androgen-unresponsive cells having more of this glycoprotein detectable on their surfaces than the androgen-responsive cells. The androgen-responsive and -unresponsive cells had similar microtubule arrays, however. During the transition from the androgen-responsive to the androgen-unresponsive phenotype, the androgen-responsive cells gradually took on the characteristics of androgen-unresponsive cells as judged by cellular morphology or the presence of focal adhesions and microfilament bundles. At intermediate stages in this process, characteristics of both cell types were visible in the cell populations. However, at the stage where all androgen-responsive characteristics were lost, the cells were no longer androgen sensitive. The loss of androgen responsiveness in Shionogi 115 mouse mammary tumor cells is correlated with changes at the cell membrane and the microfilament component of the cytoskeleton.