Objective: To evaluate the effect of 6% hydroxyethyl starch (HES) solution, with a molecular weight of 130 kDa and a degree of substitution of 0.42, on canine platelet function in vitro. Samples: Blood samples from 31 healthy adult dogs. Procedures: Citrated blood was diluted with saline (0.9% NaCl) solution or HES 130/0.42 in ratios of 1:9 (ie, 1 part saline solution or HES 130/0.42 and 9 parts blood) and 1:3. Platelet plug formation time (closure time [Ct]) was measured with a platelet function analyzer and cartridges coated with collagen and ADP. Results: Median baseline Ct with citrated blood was 84.0 seconds (interquartile range, 74.5 to 99.5 seconds). Results obtained with 1:9 dilutions with saline solution and HES 130/0.42 were not significantly different from baseline results. The 1:3 dilutions with saline solution and HES 130/0.42 resulted in median Cts of 96.0 seconds (interquartile range, 85.5 to 110.8 seconds) and 112.0 seconds (92.0 to 126.0 seconds), respectively. Results obtained with both 1:3 dilutions were significantly different from baseline results. The Ct obtained with the HES dilution was also significantly different from that of the 1:3 dilution with saline solution. Conclusions and Clinical Relevance: Saline solution and HES 130/0.42 in a 1:3 dilution affected canine platelet function by prolonging Cts. The HES 130/0.42 had a significantly greater effect on canine platelets than did saline solution.