The molecular chaperones αA- and αB-crystallins are important for cell survival and genomic stability and associate with the tubulin cytoskeleton. The mitotic spindle is abnormally assembled in a number of αA-/- and αB-/- lens epithelial cells. However, no report to date has studied the effect of α-crystallin expression on tubulin/microtubule assembly in lens epithelial cells. In the current work we tested the hypothesis that the absence of αA- and αB-crystallins alters microtubule assembly. Microtubules were reconstituted from freshly dissected explants of wildtype, αA-/-, αB-/-, and α(A/B) -/- (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. The wild-type microtubules were 4 μm long and ∼25 nm wide and had a characteristic protofilament structure, but αB-/- microtubules were 2.5-fold longer. Microtubule-associated proteins (MAPs) extracted from microtubules by washing with salt included transketolase, α-enolase, and βB2-crystallin. In DKO lens epithelial microtubules but not in wild-type, αA-/- or αB-/- microtubules, extraction of the MAPs gave very long (14-20 μm) "polyfilament" assemblies that were tightly bundled. Addition of exogenous α-crystallin (αA+ αB) was ineffective in preventing polyfilament formation. However, normal microtubule structure could be restored by including MAPs derived from wild-type lens epithelial cells during microtubule reconstitution. Intriguingly, these data suggest that α-crystallin may interact with MAPs to inhibit aggregation of microtubules in lens epithelial cells. Sedimentation analysis and 90° light scattering measurements showed that α-crystallin suppressed tubulin assembly in vitro. α-Crystallin did not have a strong effect on the GTPase activity of purified tubulin. SDS-PAGE analysis showed that α-crystallin prevented heat-induced aggregation of tubulin, suggesting that α-crystallin may affect microtubule assembly by maintaining the pool of unassembled tubulin.