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In Arabidopsis, the actin gene family comprises eight expressed and two non-expressed <i>ACTIN</i> (<i>ACT</i>) genes. Of the eight expressed isoforms, <i>ACT2</i>, <i>ACT7,</i> and <i>ACT8</i> are differentially expressed in vegetative tissues and may perform specific roles in development. Using tobacco mesophyll protoplasts, we previously demonstrated that actin-dependent clustering of chloroplasts around the nucleus prior to cell division ensures unbiased chloroplast inheritance. Here, we report that actin-dependent chloroplast clustering in Arabidopsis mesophyll protoplasts is defective in <i>act7</i> mutants, but not <i>act2-1</i> or <i>act8-2</i>. <i>ACT7</i> expression was upregulated during protoplast culture whereas <i>ACT2</i> and <i>ACT8</i> expression did not substantially change. In <i>act2-1</i>, <i>ACT7</i> expression increased in response to loss of <i>ACT2</i>, whereas in <i>act7-1</i>, neither <i>ACT2</i> nor <i>ACT8</i> expression changed appreciably in response to the absence of ACT7. Semi-quantitative immunoblotting revealed increased actin concentrations during culture, although total actin in <i>act7-1</i> was only two-thirds that of wild-type or <i>act2-1</i> after 96 h culture. Over-expression of <i>ACT2</i> and <i>ACT8</i> under control of <i>ACT7</i> regulatory sequences restored normal levels of chloroplast clustering. These results are consistent with a requirement for ACT7 in actin-dependent chloroplast clustering due to reduced levels of actin protein and gene induction in <i>act7</i> mutants, rather than strong functional specialization of the ACT7 isoform.