Interspecies hybrid cells were formed by the fusion of two parent cells: 1) the human lung cancer (bronchioloalveolar) line A549/8, which is not contact inhibited, rapidly produces tumors in athymic nude (nu/nu) mice, and forms colonies in agarose, and 2) the mouse fibroblast line 3T3-4E, which is contact inhibited, is nontumorgenic in nuce mice, and does not form colonies in agarose. These hybrid cells were tested 40-50 generations after fusion. The presence of 20 of the 23 different human chromosomes was tested by isoenzyme analysis, and examples of expression of each isoenzyme marker were found in at least some hybrid clones. All 14 independent hybrid clones tested were nontumorigenic in nude mice. Testing of hybrid clones for their ability to form colonies in agarose revealed two distinct phenotypes: agarose (clones forming colonies at 1-4% of the plated cells) and agarose (no colonies formed/10(5) cells tested). These phenotypes were discordant with all human isoenzymes tested. Malignant human lung cancer A549/8 times non-malignant mouse 3T3-4E cell hybrids were nontumorigenic in nude mice; thus malignancy of the bronchioloalveolar lung cancer behaved as a recessive trait. This nontumorigenicity was not accounted for by an absolute loss of the human chromosomes tested, but gene dosage may play a role. In contrast, the ability to clone in agarose was expressed in some hybrids (and thus behaved as a dominant trait); at present, agarose clonability cannot be related to specific human chromosomes.