Pulmonary surfactant reduces surface tension in lung alveoli and enables inspiration. Lack of surfactant can lead to infant respiratory distress syndrome and can play a role in pulmonary fibrosis. Surfactant is secreted by alveolar type II (ATII) cells in the process of exocytosis where a secretory vesicle fuses with the plasma membrane to release vesicle content. Surfactant extrusion from fused vesicles is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening (Miklavc et al., 2015). Actin coat compression allows hydrophobic surfactant to be released from the vesicle (Kittelberger et al., 2016). Emerging evidence suggests that actin can interact with microtubules; however, the interplay between actin and microtubules in exocytosis is not understood. Here we study the interaction between actin and microtubules during the post-fusion stage of exocytosis using high-resolution fluorescence microscopy on primary ATII cells. ATII cells were isolated from the lungs of male Sprague-Dawley rats according to the procedure of Dobbs et al. (1986) with minor modifications (Miklavc et al., 2010). Isolated ATII cells were transfected with actin-GFP or actin-DsRed to visualize actin coats on fused vesicles and exocytosis was stimulated with ATP (100 µM). Microtubules were localized close to actin coats and stayed close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the duration of actin coat formation and the extent of actin polymerisation on fused vesicles. Mean +/- SEM time of the peak fluorescence was 75.3 +/- 7.5 s after fusion for control, 112.4 +/- 6.8 s for colchicine and 103 +/- 6.9 s for nocodazole (p<0.05; one-way ANOVA). In actin-GFP transfected cells actin coat fluorescence intensity increase was significantly lower in control (14.48+/-1.22%; n=50 actin coats) than in cells treated with colchicine (23.82+/-1.60%; n=58; p<0.0001) or nocodazole (20.03+/-1.35%; n=56; p=0.003; two-tailed t-test). We also found that microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles. The relative intensity of actin coat fluorescence in cells transfected with IQGAP1 siRNA (14.62 +/- 1.17%; n=36 actin coats) was significantly lower than actin coat fluorescence intensity in cells treated with control siRNA (26.12 +/- 1.93%; n=37; p<0.0001; two-tailed t-test). These findings indicate that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis. This interaction may be mediated by microtubule and actin cross-linking proteins such as IQGAP1.