Primary tumors from 39 patients with non‐small cell lung carcinoma (NSCLC) were examined for cytogenetic abnormalities by conventional short‐term harvest (1–39 days) of primary cultures of minced solid‐tumor tissues and by harvest of monolayer cultures of tumor tissue (6 days to 5 months) on murine fibroblast feeder layers. A successful karyotype was obtained with both methods in nine of 39 cases. Among the remaining 30 cases, a successful karyotype was obtained in eight cases by the conventional method only and in three cases by the feeder cell method only. The success rates were 44% for the conventional method, and 31% for the feeder cell method, and the combined success rate was 51% for one or the other method. The feeder culture method, in which harvests were usually performed at later times than with the conventional method, generally produced metaphases with superior banding, which allowed clearer definition of cytogenetic abnormalities. In addition, cell lines were established in eight of these cases by the feeder cell method. Karyotypes from the longer‐term harvests typically were very similar to those from short‐term conventional cultures. Minor numerical differences and/or a few additional structural abnormalities were noted in seven of the nine cases analyzed by both methods. Overall, however, even in karyotypes from 5‐month cultures, the prominent recurrent changes and modal chromosome numbers observed in short‐term cultures were still present. The results indicate that long‐term culture with fibroblast feeder cells is a valid means of obtaining cells from solid lung tumors for cytogenetic and molecular analysis. Cell lines established by this method will be useful in future molecular studies, for example, for mapping of chromosome breakpoints and sites of chromosome loss, for in situ hybridization, and for studies of the expression of critical candidate genes implicated by cytogenetic findings. In addition, by combination of conventional and feeder cell harvests, both the number of primary tumors successfully examined karyotypically and the quality of the analyses are improved.© 1993 Wiley‐Liss, Inc.