Cytoplasmic dynein contains a series of accessory proteins associated with the motor containing heavy chains.1 These include three distinct classes of light chains (Mr < approximately 22 000). Here we demonstrate that a previously cloned protein termed rp3 is a bona fide Mr 14 000 light chain component of this microtubule motor complex. The rp3 polypeptide is approximately 55% identical to the Tctex1 dynein light chain, and together, these two proteins define one branch of a diverse family of Mr 14 000 light chains associated with both cytoplasmic and flagellar dyneins. The Tctex1 and rp3 light chains are differentially expressed in various tissues: rp3 is most prevalent in liver and brain cytoplasmic dynein, whereas those tissues contain the least amounts of Tctex1. Immunofluorescence analysis was consistent with the tissue-specific distribution of these proteins and revealed that both rp3 and Tctex1 are present in multiple perinuclear punctate particles. Furthermore, in two cell lines, rp3 was found associated with an elongated structure located in the layer of cytoplasm above the nucleus. Electrophoretic/immunological analysis indicates that there are only single isoforms for these proteins in brain and PC-12 cells, suggesting that alterations in the Mr 14 000 light chains of dynein are achieved at the level of the individual proteins and not by posttranslational modification. Dissection of the cytoplasmic dynein complex revealed that Tctex1, an Mr 8000 LC dimer, and IC74 associate to define a basal-located intermediate chain/light chain complex analogous to that found in flagellar outer arm dynein.