Chinese hamster ovary (CHO) cells were analyzed for their ability to reassemble microfilament bundles, to remain attached to a tissue culture surface, or to initiate and complete attachment onto a substrate after heat shock (45 degrees C/10 min). The cells remained attached to the tissue culture surface during and after the heat shock while the actin microfilament bundles were reversibly disrupted. Heat shock inhibited the ability of the cells to initiate and complete attachment onto a new tissue culture surface or onto a plastic surface coated with vitronectin. An inspection of the proteins present in substrate-attached material (SAM) revealed 11 major proteins containing glucosamine whose apparent Mr values were 250,000, 200,000, 150,000, 140,000, 90,000, 86,000, 82,000, 68,000, 54,000, 47,000, and 46,000. Three of the proteins (p200, p150, and p46) bound to wheat germ agglutinin while p150 and p140 bound to concanavalin A. The composition of the 11 proteins of the SAM fraction synthesized previous to the heat shock was not altered during heat shock. However, the appearance of the newly synthesized proteins in the SAM fraction was delayed by heat shock (0.5 h for p150 and 6 h for p82). The ability of heat-shocked cells to reattach onto a vitronectin-coated surface correlated with the appearance of newly synthesized p150 and p82 in the SAM fraction. Our results suggest that in addition to the microfilament bundles, heat shock may reversibly disrupt the cellular adhesion site. Further, p150 and p82, proteins whose appearance in the SAM fraction is delayed by heat shock, may be involved in the cellular attachment onto substrates, including vitronectin.