Abstract Gold(I) phosphine complexes have shown promising results as a novel class of anticancer drugs in a variety of biochemical and pharmacological investigations. Studies on pharmacokinetic properties of these species are rare. Here we report the results of a comparative study on the cytotoxicity, cellular and nuclear uptake of a series of chloro gold(I) phosphine complexes (Cl–Au–P(R) 3, R = Me, Et, tert-But, Ph) containing different ligands on the phosphor. Cellular and nuclear gold levels in HT-29 colon carcinoma and MCF-7 breast cancer cells were measured by electrothermal atomic absorption spectrometry. All studied complexes exhibited significant antiproliferative effects in both investigated cell lines. Cellular and nuclear gold levels were enhanced especially for Cl–Au–P(Ph) 3 indicating a positive influence of larger and more lipophilic substituents.